Fused amino pyridines for the treatment of brain tumors

ABSTRACT

The present invention relates to the use of compounds with fused amino pyridine core for the treatment of malignancies associated with brain and lung. The oral administration of compounds of the instant application results in effective brain penetration and provides for non-intrusive treatment of brain and lung tumors.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/688,312, filed Jan. 15, 2010, which claims the benefit of U.S.Provisional Application Nos. 61/145,297, filed Jan. 16, 2009, and61/150,402, filed Feb. 6, 2009. The entire teachings of the aboveapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Despite scientific advances in understanding the causes and treatment ofhuman malignancy, a persistent challenge facing basic and clinicalinvestigators is how to adequately treat primary and metastatic braintumors. The blood-brain barrier is a physiologic obstruction to thedelivery of systemic chemotherapy to the brain parenchyma and centralnervous system (CNS). (Deeken et al., Clin Cancer Res 2007; 13(6) 2007,1663-1674). Brain tumors are protected from systemic chemotherapy by theblood-brain barrier (BBB) and by intrinsic properties of the tumors.Pharmacologic studies of delivery of conventional chemotherapeutics andnovel therapeutics showing actual tumor concentrations and biologiceffect are lacking. (Muldoon et al. J Clin Oncol. 2007, 25(16):2295-305.Glioblastoma is the most frequent and most malignant human brain tumor.The prognosis remains very poor, with most patients dying within 1 yearafter diagnosis. (Ohgaki et al. American Journal of Pathology, 170(5),2007, 1445-1453). Thus, there remains a need to develop treatments forbrain related disorders that can cross the blood brain barrier.

Patients with secondary brain tumors also have poor treatment prognosisdue to the difficulty in delivering drugs across the blood brainbarrier. Metastatic brain tumors are the most common intracranialneoplasm in adults, and although the exact incidence is unknown, it hasbeen estimated to be as high as 200,000 cases per year in the U.S.alone. The frequency of metastatic brain tumors appears to be rising asa result of superior imaging modalities and earlier detection as well aslonger survival after a primary cancer diagnosis because of moreeffective treatment of systemic disease. (Eichler et al. The Oncologist,12 (7), 884-898, 2007).

Patients with lung cancer account for approximately 50% of brainmetastasis cases. The majority of active cytotoxic agents (like taxanes)in lung cancer treatment, are unable to effectively penetrate bloodbrain barrier (BBB). (Zarogoulidis et al., Journal of Clinical Oncology,2006 ASCO Annual Meeting Proceedings Part I, 24(18S) 2006. Consideringthe challenge in BBB penetration and the brain metastasis of lung canceranti-cancer agents that can highly distribute to lung and cross bloodbrain barriers are highly sought after in cancer treatment of braincancer and lung cancer with brain metastasis.

HSP90s are ubiquitous chaperone proteins that are involved in properprotein folding and stabilization of a wide range of proteins, includingkey proteins involved in signal transduction, cell cycle control andtranscriptional regulation. Researchers have reported that HSP90chaperone proteins are associated with important signaling proteins,such as steroid hormone receptors and protein kinases, including, e.g.,Raf-1, EGFR, v-Src family kinases, Cdk4, and ErbB-2, many of which areoverexpressed or mutated in various cancers (Buchner J. TIBS, 1999, 24,136 141; Stepanova, L. et al. Genes Dev. 1996, 10, 1491 502; Dai, K. etal. J. Biol. Chem. 1996, 271, 22030-4). Studies further indicate thatcertain co-chaperones, e.g., HSP70, p60/Hop/Stil, Hip, Bagl,HSP40/Hdj2/Hsj 1, immunophilins, p23, and p50, may assist HSP90 in itsfunction (Caplan, A. Trends in Cell Biol. 1999, 9, 262 68). HSP90 isoverexpressed in many cancers and has become a target for cancertherapy. HSP90 inhibitors possess potent anti-proliferative activity,usually at low nanomolar ranges, owing to their pharmacologicalcharacteristics of binding tightly to heat shock protein 90, coupledwith a slow dissociation rate. (Newcomb et al. Anticancer Drugs 200718(8):875-82). HSP90 has been shown to be present in a variety ofprimary and metastatic intracranial tumors including glioblastomas andmedulloblastomas (Kato et al., Acta Neuropathol. 1995; 89(2):184-8).

Recent studies also suggest that heat shock proteins (HSPs) play animportant role in neurodegenerative disorders such as Parkinson'sdisease (PD), Alzheimer's disease (AD), amyotropic lateral sclerosis(ALS), Huntington disease (HD) (Luo, G- R. Int. J. Biol. Sci., 2007,3(1), 20-26; Dickey, C., J. Clin. Invest., 2007, 117(3), p. 648-658). Ithas been shown that manipulation of HSPs, such as down regulation ofHSP90 or up regulation of HSP70, affords beneficial effects in severalneurodegenerative disorders either by reducing protein aggregation orfacilitating proper folding of proteins to restore their function.Neurodegenerative diseases such as Alzheimer's disease (AD) andHuntington's disease (polyglutamine disease) are typical diseases likelycaused by the abnormal accumulation of misfolded and aggregatedproteins, and these diseases are thought to be inhibited by the actionof Hsp70 as a chaperone. Apoptosis is one of the ways neurons die afterischemia. It has been shown that overexpression of Hsp70 in hippocampalCA1 neurons reduces evidence of protein aggregation under conditionswhere neuronal survival is increased (Giffard, R. G., et al., J. Exp.Biol. 207:3213-3220 (2004)).

A growing body of evidence supports the hypothesis that HSP90 inhibitionaffords neuroprotection in various animal models of neurologicaldisease. HSP90 has been shown by mutational analysis to be necessary forthe survival of normal eukaryotic cells. However, HSP90 is overexpressedin many tumor types indicating that it may play a significant role inthe survival of cancer cells and that cancer cells may be more sensitiveto inhibition of HSP90 than normal cells. For example, cancer cellstypically have a large number of mutated and over expressed oncoproteinsthat are dependent on HSP90 for folding. In addition, because theenvironment of a tumor is typically hostile due to hypoxia, nutrientdeprivation, acidosis, etc., tumor cells may be especially dependent onHSP90 for survival. Moreover, inhibition of HSP90 causes simultaneousinhibition of a number of client oncoproteins, as well as hormonereceptors and transcription factors making it an attractive target foran anti-cancer agent. In fact, benzoquinone ansamycins, a family ofnatural products that inhibit HSP90, has shown evidence of therapeuticactivity in clinical trials. Several promising ansamycin related HSP90inhibitors are currently in clinical trial namely, 17-allylamino17-demethoxygeldanamycin (17-AAG),17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) andIPI-504. Another class of the HSP90 inhibitor is the synthetic smallmolecule purine-scaffold. Currently, many of the purine-scaffold HSP90inhibitors are showing positive preclinical results; with the frontrunner being CNF-2024, which is currently in phase 1 clinical trial.

In recent years, molecularly targeted therapies, such as epidermalgrowth factor receptor (EGFR) inhibitors, have gained tremendousattention for their potential to improve patient survival and reducetoxic side effects, in particular for the treatment of lung cancer. Yet,early clinical trials of these inhibitors, such as gefitinib anderlotinib, were modestly encouraging, with a response in only ˜10% ofpatients who carry genetic mutations of EGFR (Bao et al., Mol. Cancer.Ther. 2009; 8(12) 2009). In addition, resistance almost invariablydevelops in these non-small cell lung cancer (NSCLC) patients althoughthey respond to these receptor tyrosine kinase (RTK) inhibitorsinitially. Of these instances of so-called “acquired” resistance, it isestimated that ˜50% are due to the emergence of an additional EGFRmutation in exon 20 (EGFRT790M), the “gatekeeper” residue within thekinase domain (Kobayashi et al., N. Engl. J. Med. 2005; 352: 786-92;Proc. Natl. Acad. Sci. 2005, 102 11011-6). Structural analysis suggeststhat the T790M mutation sterically hinders the binding of erlotinib tothe EGFR kinase domain by introducing a bulky methionine residue,thereby conferring erlotinib resistance (8, 9). There is also evidenceto suggest that T790M mutation causes drug resistance by increasing theaffinity of EGFR for ATP (Yun et al., Proc. Natl. Acad. Sci. 2008, 105,2070-5). To overcome such EGFRT790M-mediated resistance, severalirreversible EGFR inhibitors able to form covalent bonds with Cys-797 atthe edge of the ATP binding site are actively being tested in clinicaltrials. However, only modest efficacy has been reported, believed to bein part due to persistent PI3K/AKT/mTOR signaling following treatment(Bao et al.).

Drugs targeting the protein HSP90 are quite new in cancer andneurodegenerative disease therapies. Their presence in many of thetumors associated with CNS point to a need for HSP-related drugs capableof crossing the blood brain barrier. As such, a promising therapy forbrain related disorders would be HSP90 inhibitors that are efficient incrossing the blood brain barrier. This invention relates to fused aminopyridine compounds useful as HSP90 inhibitors for the treatment of brainrelated disorders. This invention further relates to treatment ofcancers that are resistant to other epidermal growth factor receptorinhibitors. This invention further relates to the inhibition of HSP70and the treatment of diseases related to HSP70.

SUMMARY OF THE INVENTION

The present invention relates to the unexpected discovery that certainHSP90 inhibitors containing fused amino pyridine core have good toexcellent brain tissue deposition. The discovery supports the use ofsuch compounds in the treatment of HSP90 related diseases and disorderssuch as cancer associated with brain and lung.

Accordingly, the present invention provides a method of treating brainand lung related disorders using a compound having the general formulaI:

or its pharmaceutically acceptable salts thereof, wherein;

-   -   n is 1 or 2;    -   R₁ and R₂ are independently H, C₁-C₈ alkyl, C₁-C₈ substituted        alkyl;    -   R₃ and R₄ are independently H, C₁-C₈ alkyl, C₁-C₈ substituted        alkyl; R₅ is halogen, —SR₆ or —NR₆R₇ wherein R₆ and R₇ are        independently H, C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₂-C₈        alkenyl, C₂-C₈ substituted alkenyl, C₂-C₈ alkynyl, C₂-C₈        substituted alkynyl or C₃-C₈ cycloalkyl; and,    -   Y is C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₂-C₈ alkenyl, C₂-C₈        substituted alkenyl, C₂-C₈ alkynyl, C₂-C₈ substituted alkynyl,        C₃-C₈ cycloalkyl or C₃-C₈ substituted cycloalkyl.

The invention also relates to the use of such compounds in themanufacture of a medicament for the treatment of brain relateddisorders, such as glioblastoma multiforme, neurodegenerative diseasessuch as Alzheimer's disease, and lung related disorders such as smallcell and non-small cell lung carcinomas.

DETAILED DESCRIPTION OF THE INVENTION

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1A: Plasma concentrations of compounds 2 and 15 in tumor-bearingmice after IV administration (5 mg/kg), obtained through a cassettedosing study.

FIG. 1B: Plasma concentrations of compounds 2 and 15 in tumor-bearingmice after oral (10 mg/kg) administration, obtained through a cassettedosing study.

FIG. 1C: Plasma pharmacokinetic values for compounds 2 and 15 after IV(5 mg/kg) and Oral (10 mg/kg) administration.

FIG. 2A: Tumor concentrations of compounds 2 and 15 in tumor-bearingmice after IV (5 mg/kg) administration, obtained through a cassettedosing study.

FIG. 2B: Tumor concentrations of compounds 2 and 15 in tumor-bearingmice after oral (10 mg/kg) administration, obtained through a cassettedosing study.

FIG. 2C: Plasma pharmacokinetic values for compounds 2 and 15 after IV(5 mg/kg) and Oral (10 mg/kg) administration.

FIG. 3A: Brain concentrations of compounds 2 and 15 in tumor-bearingmice after IV (5 mg/kg).

FIG. 3B: Brain concentrations of compounds 2 and 15 in tumor-bearingmice after oral administration (10 mg/kg).

FIG. 3C: Brain AUC values for compounds 2 and 15 after IV (5 mg/kg) andOral (10 mg/kg) administration.

FIG. 4: Tissue concentrations of compound 15 in (a) plasma, (b) tumor,(c) brain, and (d) lung after oral administration (30 mg/kg), obtainedthrough a single dose study; (e) Pharmacokinetic profile of compound 15in plasma, tumor, brain and lung tissue after oral administration (30mg/kg).

FIG. 5: (a) Plasma concentration and (b) pharmacokinetic profile ofcompound 15 after administration to tumor bearing mice in 10 mg/kg IV,30 mg/kg p.o. and 160 mg/kg p.o. dosages showing bioavailability andhalf-life in plasma.

FIG. 6: (a) Tumor concentration and (b) pharmacokinetic profile ofcompound 15 after administration to tumor bearing mice in 10 mg/kg IV,30 mg/kg p.o. and 160 mg/kg p.o. dosages showing bioavailability andhalf-life in tumor.

FIG. 7: Lung concentration of compound 15 after oral administration (10mg/kg).

FIG. 8A: Plasma and tissue concentrations of Compound 2 after IV (5gm/Kg) administration.

FIG. 8B: Plasma and tissue concentrations of Compound 2 after oral (10gm/Kg) administration.

FIG. 9: Comparison of brain penetration of compounds 2 and 21 withreference compounds (VER-052296, Cmp42, SNX-2112) after IV (5 mg/Kg)administration.

FIG. 10: Comparison of brain penetration of compounds 2 and 21 withreference compounds (VER-052296, Cmp42, SNX-2112) after oral (10 mg/Kg)administration.

FIG. 11: Efficacy Study of compound-2 in Rat tMCAO Model (Single IVDose, 4 hrs Post Occlusion).

FIG. 12: Efficacy Study of compound-2 in Rat tMCAO Model (Single IV Dose(2.5 mg/Kg or 5 mg/Kg), 4 hrs Post Occlusion).

FIG. 13: Compound 21 induces HSP70 up-regulation in primary hippocampalneuron culture.

FIG. 14: Compound 21 decreases PHF-tau level in primary hippocampalneuron cultures.

FIG. 15A: Comparison of control (C, DMSO treated) and Compound-15treated (T) H1993 NSCLC cells. Results indicate that phosphorylated andtotal EGFR, MET, AKT, and MAPK (ERK) are durably inhibited by ashort-term treatment with Compound-15.

FIG. 15B: Comparison of control (C, DMSO treated) and Compound-15treated (T) H1975 NSCLC cells. Results indicate that phosphorylated andtotal EGFR, MET, AKT, and MAPK (ERK) are durably inhibited by ashort-term treatment with Compound-15.

FIG. 16: Fluorescence polarization competition binding assays were donewith H1975 and H1993 NSCLC cancer cell extracts with competition fromfluorescein isothiocyanate-labeled geldanamycin in the presence ofvarying concentrations of Compound-15.

FIG. 17: Inhibition of HSP90 Client Proteins by Compound-15.

FIG. 18A: Efficacy study of Compound-15 in the H1975 NSCLC subcutaneoustumor model.

FIG. 18B: Animal body weight changes relative to pretreatment values inan efficacy study in the H1975 NSCLC subcutaneous tumor model (n=8).

FIG. 19A: Efficacy study of Compound-15 in H1975 NSCLC orthotopic lungtumor model in comparison with erlotinib.

FIG. 19B: Dose-dependent efficacy of Compound-15 in H1975 NSCLCorthotopic lung tumor model.

FIG. 19C: Pharmacodynamic study of Compound-15 in H1975 NSCLC orthotopiclung tumor model.

FIG. 19D: Efficacy study of Compound-15 in H1975 NSCLC orthotopic lungtumor model in comparison with lapatinib.

FIG. 20A: Pharmacodynamic study in K-ras-mutated A549 (human lungadenocarcinoma epithelial cell line) subcutaneous tumors.

FIG. 20B: Efficacy study of Compound-15 in A549 subcutaneous tumormodel.

FIG. 20C: Efficacy study in A549 orthotopic lung tumor model.

FIG. 20D: Efficacy study of Compound-15 in combination with paclitaxelin A549 orthotopic lung tumor model.

FIG. 21A: Tumor growth after oral delivery of Compound 15 in variousdoses.

FIG. 21B: Pharmacodynamic analysis (Western Blot) of tumors in micetreated with 40, 80 or 160 mg/kg (q2d).

FIG. 22A: Efficacy study of Compound-15 in MV4-11 (human lymphoblastcell line) s.c. tumor model.

FIG. 22B: Efficacy study of Compound-15 in MV4-11 s.c. tumor model withpretreatment tumor volume of 146 mm³.

FIG. 22C: Efficacy study of Compound-15 in MV4-11 s.c. tumor model withpretreatment tumor volume of 380 mm³.

FIG. 22D: Efficacy study of Compound-15 in MV4-11 s.c. tumor model withpretreatment tumor volume of 835 mm³.

FIG. 23A: Efficacy study of Compound-15 in H1975 NSCLC s.c. tumor model.

FIG. 23B: Efficacy study of Compound-15 in H1975 NSCLC s.c. tumor model.

FIG. 23C: Efficacy study of Compound-15 in combination with paclitaxelin H1975 NSCLC s.c. tumor model.

FIG. 23D: Efficacy study of Compound-15 in combination with camptothecinin H1975 NSCLC s.c. tumor model.

The instant invention relates to the use of compounds of Formula I forthe treatment of brain related disorders including brain tumors,malignancies, metastases and neurodegenerative diseases, as well as forthe treatment of lung related disorders including neoplastic lungdisorders such as non-small cell lung cancer. Compounds of the formula Ihave been described in the co-pending application Ser. No. 12/045,509(US Patent Application Publication No. 20080234314 A1) and 61/015,288,the entire contents of which are incorporated by reference herein. Afirst embodiment is the use of the compounds represented by formula (I)as illustrated above, or its geometric isomers, enantiomers,diastereomers, racemates, pharmaceutically acceptable salts, prodrugsand solvates thereof for the treatment of brain related disorders.

Representative compounds according to the invention are in the Table Abelow or pharmaceutically acceptable salts thereof. Preferred compoundsaccording to the invention are compounds 2, 15 and 21.

TABLE A Compound # Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

Compounds of formula I with a c Log P value of about 3.70 or more areconsidered more effective in crossing the blood brain barrier and, areaccordingly, preferred. Preferred compounds have a c Log P value of atleast 4.00 and most preferred compounds have a c Log P value of at least4.20.

The invention further relates to a method of regulating the level ofHSP70 in the brain tissue of patient by the oral administration of acompound of formula I to a patient in need thereof.

The invention further relates to a method of treating lung cancer in apatient that fails to respond to treatment by an inhibitor of epidermalgrowth factor (EGFR). In particular the invention relates to thetreatment of lung cancers that have grown resistant to an EGFR inhibitorselected from gefitinib, erlotinib, vandetanib, AEE-788, PKI-166,PTK787/ZK222584, lapatinib, cetuximab, nimotuzumab, matuzumab,panitumumab, trastuzumab and pertuzumab by administration of a compoundof formula I. In one embodiment the failure of the EGFR inhibitorsrefers to decreased effectiveness of an EGFR inhibitor in a patient dueto EGFR gene mutations. In one embodiment, the resistance is acquiredresistance due to a gene mutation, for example, T790M or D761Y or L858Rmutation of the EGFR gene. In one embodiment, the resistance is aprimary resistance due to a gene mutation, for example, K-Ras genemutation.

The term “cancer” refers to any cancer caused by the proliferation ofmalignant neoplastic cells, such as tumors, neoplasms, carcinomas,sarcomas, leukemias, lymphomas and the like. The present invention isrelated to treating CNS or brain disorders, in particular cancers of thebrain, such as brain tumors (including gliomas such as ependymomas,astrocytomas, gangliogliomas, oligodendrogliomas and glioblastomas),pituitary tumors, neuroblastoma, retinoblastoma, medulloblastoma, andmeningioma, and also include metastases of primary tumors from anoriginal site outside of the brain or CNS. The present invention isrelated to treating lung related disorders such as small cell lunchcancer, non-small cell lung cancers including adenocarcinoma, squamouscell lung carcinoma, large cell lung carcinoma.

It will be appreciated that compound of the inventions can be useful intreating “neurodegenerative diseases” including Huntington's disease,Polyglutamine disease, Parkinson's disease, Alzheimer's disease,Seizures, Striatonigral degeneration, Progressive supranuclear palsy,Torsion dystonia, Spasmodic torticollis and dyskinesis, Familial tremor,Gilles de la Tourette syndrome, Diffuse Lewy body disease, Progressivesupranuclear palsy, Pick's disease, intracerebral hemorrhage, Primarylateral sclerosis, Spinal muscular atrophy, Amyotrophic lateralsclerosis, Hypertrophic interstitial polyneuropathy, Hereditary spasticparaplegia, Progressive ataxia and Shy-Drager syndrome.

In one embodiment, the present invention includes the use of one or morecompounds of the invention in the manufacture of a medicament thatprevents further aberrant proliferation, differentiation, or survival ofcells. For example, compounds of the invention may be useful inpreventing tumors from increasing in size or from reaching a metastaticstate. The subject compounds may be administered to halt the progressionor advancement of cancer. In addition, the instant invention includesuse of the subject compounds to prevent a recurrence of cancer.

“Combination therapy” includes the administration of the subjectcompounds in further combination with other biologically activeingredients (such as, but not limited to, a second and differentantineoplastic agent) and non-drug therapies (such as, but not limitedto, surgery or radiation treatment). For instance, the compounds of theinvention can be used in combination with other pharmaceutically activecompounds, preferably compounds that are able to enhance the effect ofthe compounds of the invention. The compounds of the invention can beadministered simultaneously (as a single preparation or separatepreparation) or sequentially to the other drug therapy. In general, acombination therapy envisions administration of two or more drugs duringa single cycle or course of therapy.

In one aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate protein kinases involved in various disease states. Examples ofsuch kinases may include, but are not limited to: serine/threoninespecific kinases, receptor tyrosine specific kinases and non-receptortyrosine specific kinases. Serine/threonine kinases include mitogenactivated protein kinases (MAPK), meiosis specific kinase (MEK), RAF andaurora kinase. Examples of receptor kinase families include epidermalgrowth factor receptor (EGFR) (e.g. HER2/neu, HER3, HER4, ErbB, ErbB2,ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor(e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R);hepatocyte growth/scatter factor receptor (HGFR) (e.g, MET, RON, SEA,SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK,EEK, EHK-1, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); Axl (e.g.Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR)(e.g. PDGFα-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1,FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but arenot limited to, BCR-ABL (e.g. p43^(abl), ARG); BTK (e.g. ITK/EMT, TEC);CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate non-kinase biological targets or processes. Such targetsinclude histone deacetylases (HDAC), DNA methyltransferase (DNMT), andproteosomes.

In a preferred embodiment, subject compounds may be combined withantineoplastic agents (e.g. small molecules, monoclonal antibodies,antisense RNA, and fusion proteins) that inhibit one or more biologicaltargets such as Zolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent,Sprycel, Nexavar, Sorafinib, CNF2024, RG108, BMS387032, Affinitak,Avastin, Herceptin, Erbitux, AG24322, PD325901, ZD6474, PD184322,Obatodax, ABT737 and AEE788. Such combinations may enhance therapeuticefficacy over efficacy achieved by any of the agents alone and mayprevent or delay the appearance of resistant mutational variants.

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents encompass a wide range of therapeutic treatmentsin the field of oncology. These agents are administered at variousstages of the disease for the purposes of shrinking tumors, destroyingremaining cancer cells left over after surgery, inducing remission,maintaining remission and/or alleviating symptoms relating to the canceror its treatment. Examples of such agents include, but are not limitedto, alkylating agents such as mustard gas derivatives (Mechlorethamine,cylophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines(thiotepa, hexamethylmelanine), Alkylsulfonates (Busulfan), Hydrazinesand Triazines (Altretamine, Procarbazine, Dacarbazine and Temozolomide),Nitrosoureas (Carmustine, Lomustine and Streptozocin), Ifosfamide andmetal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloidssuch as Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxeland Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine andVinorelbine), and Camptothecan analogs (Irinotecan and Topotecan);anti-tumor antibiotics such as Chromomycins (Dactinomycin andPlicamycin), Anthracyclines (Doxorubicin, Daunorubicin, Epirubicin,Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibioticssuch as Mitomycin, Actinomycin and Bleomycin; anti-metabolites such asfolic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed,Aminopterin), pyrimidine antagonists (5-Fluorouracil, Floxuridine,Cytarabine, Capecitabine, and Gemcitabine), purine antagonists(6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors(Cladribine, Fludarabine, Mercaptopurine, Clofarabine, Thioguanine,Nelarabine and Pentostatin); topoisomerase inhibitors such astopoisomerase I inhibitors (Ironotecan, topotecan) and topoisomerase IIinhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide);monoclonal antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab,Trastuzumab, Ibritumomab Tioxetan, Cetuximab, Panitumumab, Tositumomab,Bevacizumab); and miscellaneous anti-neoplastics such as ribonucleotidereductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor(Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubuleagents (Estramustine); and retinoids (Bexarotene, Isotretinoin,Tretinoin (ATRA).

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemoprotective agent.Chemoprotective agents act to protect the body or minimize the sideeffects of chemotherapy. Examples of such agents include, but are notlimited to, amfostine, mesna, and dexrazoxane.

In one aspect of the invention, the subject compounds are administeredin combination with radiation therapy. Radiation is commonly deliveredinternally (implantation of radioactive material near cancer site) orexternally from a machine that employs photon (x-ray or gamma-ray) orparticle radiation. Where the combination therapy further comprisesradiation treatment, the radiation treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and radiation treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the radiation treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

The invention encompasses pharmaceutical compositions comprisingpharmaceutically acceptable salts of the compounds of the invention asdescribed above. Preferred salts include hydrochlorates, sulfonates,lower alkylsulfonates (including methylsulfonates), fumarates, maleates,tartrates and citrates. The invention also encompasses pharmaceuticalcompositions comprising hydrates of the compounds of the invention. Theterm “hydrate” includes but is not limited to hemihydrate, monohydrate,dihydrate, trihydrate and the like. The invention further encompassespharmaceutical compositions comprising any solid or liquid physical formof the compound of the invention. For example, the compounds can be in acrystalline form, in amorphous form, and have any particle size. Theparticles may be micronized, or may be agglomerated, particulategranules, powders, oils, oily suspensions or any other form of solid orliquid physical form.

The compounds of the invention, and derivatives, fragments, analogs,homologs, pharmaceutically acceptable salts or hydrate thereof can beincorporated into pharmaceutical compositions suitable foradministration, together with a pharmaceutically acceptable carrier orexcipient. Such compositions typically comprise a therapeuticallyeffective amount of any of the compounds above, and a pharmaceuticallyacceptable carrier. Preferably, the effective amount when treatingcancer is an amount effective to selectively induce terminaldifferentiation of suitable neoplastic cells and less than an amountwhich causes toxicity in a patient.

It will be appreciated that the compounds of the invention mayadvantageously be used in conjunction with one or more other therapeuticagents. Examples of suitable agents for adjunctive therapy include a5HT₁ agonist, such as a triptan (e.g. sumatriptan or naratriptan); anadenosine A1 agonist; an EP ligand; an NMDA modulator, such as a glycineantagonist; a sodium channel blocker (e.g. lamotrigine); a substance Pantagonist (e.g. an NKI antagonist); a cannabinoid; acetaminophen orphenacetin; a 5-lipoxygenase inhibitor; a leukotriene receptorantagonist; a DMARD (e.g. methotrexate); gabapentin and relatedcompounds; a tricyclic antidepressant (e.g. amitryptilline); a neuronestabilising antiepileptic drug; a mono-aminergic uptake inhibitor (e.g.venlafaxine); a matrix metalloproteinase inhibitor; a nitric oxidesynthase (NOS) inhibitor, such as an iNOS or an nNOS inhibitor; aninhibitor of the release, or action, of tumour necrosis factor .alpha.;an antibody therapy, such as a monoclonal antibody therapy; an antiviralagent, such as a nucleoside inhibitor (e.g. lamivudine) or an immunesystem modulator (e.g. interferon); an opioid analgesic; a localanaesthetic; a stimulant, including caffeine; an H₂-antagonist (e.g.ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g.aluminum or magnesium hydroxide; an antiflatulent (e.g. simethicone); adecongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine,oxymetazoline, epinephrine, naphazoline, xylometazoline,propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine,hydrocodone, carmiphen, carbetapentane, or dextramethorphan); adiuretic; or a sedating or non-sedating antihistamine.

In one embodiment, the pharmaceutical compositions are administeredorally, and are thus formulated in a form suitable for oraladministration, i.e., as a solid or a liquid preparation. Suitable solidoral formulations include tablets, capsules, pills, granules, pellets,sachets and effervescent, powders, and the like. Suitable liquid oralformulations include solutions, suspensions, dispersions, emulsions,oils and the like. In one embodiment of the present invention, thecomposition is formulated in a capsule. In accordance with thisembodiment, the compositions of the present invention comprise inaddition to the active compound and the inert carrier or diluent, a hardgelatin capsule.

Any inert excipient that is commonly used as a carrier or diluent may beused in the formulations of the present invention, such as for example,a gum, a starch, a sugar, a cellulosic material, an acrylate, ormixtures thereof. A preferred diluent is microcrystalline cellulose. Thecompositions may further comprise a disintegrating agent (e.g.,croscarmellose sodium) and a lubricant (e.g., magnesium stearate), andin addition may comprise one or more additives selected from a binder, abuffer, a protease inhibitor, a surfactant, a solubilizing agent, aplasticizer, an emulsifier, a stabilizing agent, a viscosity increasingagent, a sweetener, a film forming agent, or any combination thereof.Furthermore, the compositions of the present invention may be in theform of controlled release or immediate release formulations.

For liquid formulations, pharmaceutically acceptable carriers may beaqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Examples of oils arethose of petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, andfish-liver oil. Solutions or suspensions can also include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

In addition, the compositions may further comprise binders (e.g.,acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g., cornstarch, potato starch, alginic acid,silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodiumstarch glycolate, Primogel), buffers (e.g., tris-HCl., acetate,phosphate) of various pH and ionic strength, additives such as albuminor gelatin to prevent absorption to surfaces, detergents (e.g., Tween20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors,surfactants (e.g., sodium lauryl sulfate), permeation enhancers,solubilizing agents (e.g., glycerol, polyethylene glycerol), a glidant(e.g., colloidal silicon dioxide), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g.,hydroxypropyl cellulose, hydroxypropylmethyl cellulose), viscosityincreasing agents (e.g., carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citricacid), flavoring agents (e.g., peppermint, methyl salicylate, or orangeflavoring), preservatives (e.g., Thimerosal, benzyl alcohol, parabens),lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol,sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide),plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers(e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate),polymer coatings (e.g., poloxamers or poloxamines), coating and filmforming agents (e.g., ethyl cellulose, acrylates, polymethacrylates),cyclodextrins such as alpha-(α), beta-(β) and gamma-(γ)cyclodextrins,preferably beta-cyclodextrins, (e.g., hydroxypropyl beta-cyclodextrinsand sulfoalkyl ether beta-cyclodextrins (especially sulfobutyl etherbeta-cyclodextrins)) and/or adjuvants.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Daily administration may be repeated continuously for a period ofseveral days to several years. Oral treatment may continue for betweenone week and the life of the patient. Preferably the administration maytake place for five consecutive days after which time the patient can beevaluated to determine if further administration is required. Theadministration can be continuous or intermittent, e.g., treatment for anumber of consecutive days followed by a rest period or on anevery-other-day dosing schedule which may be followed by a rest period.The compounds of the present invention may be administered intravenouslyon the first day of treatment, with oral administration on the secondday and all consecutive days thereafter.

The preparation of pharmaceutical compositions that contain an activecomponent is well understood in the art, for example, by mixing,granulating, or tablet-forming processes. The active therapeuticingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the active agents are mixed with additives customary forthis purpose, such as vehicles, stabilizers, or inert diluents, andconverted by customary methods into suitable forms for administration,such as tablets, coated tablets, hard or soft gelatin capsules, aqueous,alcoholic or oily solutions and the like as detailed above.

The amount of the compound administered to the patient is less than anamount that would cause toxicity in the patient. In certain embodiments,the amount of the compound that is administered to the patient is lessthan the amount that causes a concentration of the compound in thepatient's plasma to equal or exceed the toxic level of the compound.

Preferably, the concentration of the compound in the patient's plasma ismaintained at about 10 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 25 nM. In oneembodiment, the concentration of the compound in the patient's plasma ismaintained at about 50 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 100 nM. In oneembodiment, the concentration of the compound in the patient's plasma ismaintained at about 500 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 1000 nM. In oneembodiment, the concentration of the compound in the patient's plasma ismaintained at about 2500 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 5000 nM. Theoptimal amount of the compound that should be administered to thepatient in the practice of the present invention will depend on theparticular compound used and the type of cancer being treated.

DEFINITIONS

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The term “alkyl” embraces linear or branched radicals having one toabout eight carbon atoms. More preferred alkyl radicals are “loweralkyl” radicals having one to about six carbon atoms. Examples of suchradicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl and the like.

The term “alkenyl” embraces linear or branched radicals having at leastone carbon-carbon double bond of two to about eight carbon atoms.Examples of alkenyl radicals include ethenyl, allyl, propenyl, butenyland 4-methylbutenyl. The terms “alkenyl”, and “lower alkenyl”, embraceradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations.

The term “alkynyl” embraces linear or branched radicals having at leastone carbon-carbon triple bond of two to about eight carbon atoms.Examples of alkynyl radicals include propargyl, 1-propynyl, 2-propynyl,1-butyne, 2-butynyl and 1-pentynyl.

The term “cycloalkyl” embraces saturated and unsaturated carbocyclicradicals having three to about eight carbon atoms. Examples of suchradicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclobutenyl, cyclopentenyl and cyclohexenyl.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, cyano, nitro, hydroxy,thiol, aliphatic groups (such as alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl,alkylamino, alkylaminoalkyl, and aminoalkylamino), and aromatic groups(such as arylamino, arylaminoalkyl, aryl, and heteroaryl). It isunderstood that the substituent may be further substituted. Preferably,the substituent is not an oxo or acyl group.

For simplicity, chemical moieties are defined and referred to throughoutcan be univalent chemical moieties (e.g., alkyl, aryl, etc.) ormultivalent moieties under the appropriate structural circumstancesclear to those skilled in the art. For example, an “alkyl” moiety can bereferred to a monovalent radical (e.g. CH₃—CH₂—), or in other instances,a bivalent linking moiety can be “alkyl,” in which case those skilled inthe art will understand the alkyl to be a divalent radical (e.g.,—CH₂—CH₂—), which is equivalent to the term “alkylene.”

As used herein, the term “aberrant proliferation” refers to abnormalcell growth.

The phrase “adjunctive therapy” encompasses treatment of a subject withagents that reduce or avoid side effects associated with the combinationtherapy of the present invention, including, but not limited to, thoseagents, for example, that reduce the toxic effect of anticancer drugs,e.g., bone resorption inhibitors, cardioprotective agents; prevent orreduce the incidence of nausea and vomiting associated withchemotherapy, radiotherapy or operation; or reduce the incidence ofinfection associated with the administration of myelosuppressiveanticancer drugs.

The term “cancer” as used herein denotes a class of diseases ordisorders characterized by uncontrolled division of cells and theability of these cells to invade other tissues, either by direct growthinto adjacent tissue through invasion or by implantation into distantsites by metastasis.

The term “compound” is defined herein to include pharmaceuticallyacceptable salts, solvates, hydrates, polymorphs, enantiomers,diastereoisomers, racemates and the like of the compounds having aformula as set forth herein.

The term “device” refers to any appliance, usually mechanical orelectrical, designed to perform a particular function.

The term “inhibition,” in the context of neoplasia, tumor growth ortumor cell growth, may be assessed by delayed appearance of primary orsecondary tumors, slowed development of primary or secondary tumors,decreased occurrence of primary or secondary tumors, slowed or decreasedseverity of secondary effects of disease, arrested tumor growth andregression of tumors, among others. In the extreme, complete inhibition,is referred to herein as prevention or chemoprevention.

The term “metastasis,” as used herein, refers to the migration of cancercells from the original tumor site through the blood and lymph vesselsto produce cancers in other tissues. Metastasis also is the term usedfor a secondary cancer growing at a distant site.

The term “neoplasm,” as used herein, refers to an abnormal mass oftissue that results from excessive cell division. Neoplasms may bebenign (not cancerous), or malignant (cancerous) and may also be calleda tumor. The term “neoplasia” is the pathological process that resultsin tumor formation.

As used herein, the term “pre-cancerous” refers to a condition that isnot malignant, but is likely to become malignant if left untreated.

The term “proliferation” refers to cells undergoing mitosis.

The phrase a “radio therapeutic agent” refers to the use ofelectromagnetic or particulate radiation in the treatment of neoplasia.

The term “recurrence” as used herein refers to the return of cancerafter a period of remission. This may be due to incomplete removal ofcells from the initial cancer and may occur locally (the same site ofinitial cancer), regionally (in vicinity of initial cancer, possibly inthe lymph nodes or tissue), and/or distally as a result of metastasis.

The term “treatment” refers to any process, action, application,therapy, or the like, wherein a mammal, including a human being, issubject to medical aid with the object of improving the mammal'scondition, directly or indirectly.

As used herein, the term “effective amount of the subject compounds,”with respect to the subject method of treatment, refers to an amount ofthe subject compound which, when delivered as part of desired doseregimen, brings about, e.g. a change in the rate of cell proliferationand/or state of differentiation and/or rate of survival of a cell toclinically acceptable standards. This amount may further relieve to someextent one or more of the symptoms of a neoplasia disorder, including,but is not limited to: 1) reduction in the number of cancer cells; 2)reduction in tumor size; 3) inhibition (i.e., slowing to some extent,preferably stopping) of cancer cell infiltration into peripheral organs;4) inhibition (i.e., slowing to some extent, preferably stopping) oftumor metastasis; 5) inhibition, to some extent, of tumor growth; 6)relieving or reducing to some extent one or more of the symptomsassociated with the disorder; and/or 7) relieving or reducing the sideeffects associated with the administration of anticancer agents.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting the free base function with asuitable organic acid or inorganic acid. Examples of pharmaceuticallyacceptable nontoxic acid addition salts include, but are not limited to,salts of an amino group formed with inorganic acids such as hydrochloricacid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloricacid or with organic acids such as acetic acid, maleic acid, tartaricacid, citric acid, succinic acid, lactobionic acid or malonic acid or byusing other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include, but are not limited to,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, sulfonate and aryl sulfonate.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals with undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use, aswell as the zwitterionic forms, where possible, of the compounds of thepresent invention. “Prodrug”, as used herein means a compound which isconvertible in vivo by metabolic means (e.g. by hydrolysis) to acompound of the invention. Various forms of prodrugs are known in theart, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs,Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4,Academic Press (1985); Krogsgaard-Larsen, et al., (ed.). “Design andApplication of Prodrugs, Textbook of Drug Design and Development,Chapter 5, 113-191 (1991); Bundgaard, et al., Journal of Drug DeliverReviews, 8:1-38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285et seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel DrugDelivery Systems, American Chemical Society (1975); and Bernard Testa &Joachim Mayer, “Hydrolysis In Drug And Prodrug Metabolism: Chemistry,Biochemistry And Enzymology,” John Wiley and Sons, Ltd. (2002).

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration, such as sterilepyrogen-free water. Suitable carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, a standard referencetext in the field, which is incorporated herein by reference. Preferredexamples of such carriers or diluents include, but are not limited to,water, saline, finger's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

As used herein, the term “pre-cancerous” refers to a condition that isnot malignant, but is likely to become malignant if left untreated.

The term “subject” as used herein refers to an animal. Preferably theanimal is a mammal. More preferably the mammal is a human. A subjectalso refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, fish, birds and the like.

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The compounds described herein may contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids.The present invention is meant to include all such possible isomers, aswell as their racemic and optically pure forms. Optical isomers may beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). Whenthe compounds described herein contain olefinic double bonds, otherunsaturation, or other centers of geometric asymmetry, and unlessspecified otherwise, it is intended that the compounds include both Eand Z geometric isomers and/or cis- and trans-isomers. Likewise, alltautomeric forms are also intended to be included. The configuration ofany carbon-carbon double bond appearing herein is selected forconvenience only and is not intended to designate a particularconfiguration unless the text so states; thus a carbon-carbon doublebond or carbon-heteroatom double bond depicted arbitrarily herein astrans may be cis, trans, or a mixture of the two in any proportion.

Pharmaceutical Compositions

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a compound of the present inventionformulated together with one or more pharmaceutically acceptablecarriers or excipients.

As used herein, the term “pharmaceutically acceptable carrier orexcipient” means a non-toxic, inert solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose and sucrose;cyclodextrins such as alpha-(α), beta-(β) and gamma-(γ)cyclodextrins;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols such as propylene glycol; esters such as ethyloleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or: a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

For pulmonary delivery, a therapeutic composition of the invention isformulated and administered to the patient in solid or liquidparticulate form by direct administration e.g., inhalation into therespiratory system. Solid or liquid particulate forms of the activecompound prepared for practicing the present invention include particlesof respirable size: that is, particles of a size sufficiently small topass through the mouth and larynx upon inhalation and into the bronchiand alveoli of the lungs. Delivery of aerosolized therapeutics,particularly aerosolized antibiotics, is known in the art (see, forexample U.S. Pat. No. 5,767,068 to VanDevanter et al., U.S. Pat. No.5,508,269 to Smith et al., and WO 98/43650 by Montgomery, all of whichare incorporated herein by reference). A discussion of pulmonarydelivery of antibiotics is also found in U.S. Pat. No. 6,014,969,incorporated herein by reference.

By a “therapeutically effective amount” of a compound of the inventionis meant an amount of the compound which confers a therapeutic effect onthe treated subject, at a reasonable benefit/risk ratio applicable toany medical treatment. The therapeutic effect may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). An effective amount of the compounddescribed above may range from about 0.1 mg/Kg to about 500 mg/Kg,preferably from about 1 to about 50 mg/Kg. Effective doses will alsovary depending on route of administration, as well as the possibility ofco-usage with other agents. It will be understood, however, that thetotal daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or contemporaneously with thespecific compound employed; and like factors well known in the medicalarts.

The total daily dose of the compounds of this invention administered toa human or other animal in single or in divided doses can be in amounts,for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1to 25 mg/kg body weight. Single dose compositions may contain suchamounts or submultiples thereof to make up the daily dose. In general,treatment regimens according to the present invention compriseadministration to a patient in need of such treatment from about 10 mgto about 1000 mg of the compound(s) of this invention per day in singleor multiple doses.

The compounds of the formulae described herein can, for example, beadministered by injection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, topically, in an ophthalmicpreparation, or by inhalation, with a dosage ranging from about 0.1 toabout 500 mg/kg of body weight, alternatively dosages between 1 mg and1000 mg/dose, every 4 to 120 hours, or according to the requirements ofthe particular drug. The methods herein contemplate administration of aneffective amount of compound or compound composition to achieve thedesired or stated effect. Typically, the pharmaceutical compositions ofthis invention will be administered from about 1 to about 6 times perday or alternatively, as a continuous infusion. Such administration canbe used as a chronic or acute therapy. The amount of active ingredientthat may be combined with pharmaceutically excipients or carriers toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. A typical preparation willcontain from about 5% to about 95% active compound (w/w). Alternatively,such preparations may contain from about 20% to about 80% activecompound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Patients may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

Synthetic Methods

The compounds of the invention, or a pharmaceutically-acceptable saltthereof, may be prepared by any process known to be applicable to thepreparation of chemically-related compounds. Suitable processes formaking certain intermediates include, for example, those illustrated inPCT publication numbers WO 2008/115719, WO 2002/36075, WO 2003/037860and WO 2006/084030. Necessary starting materials may be obtained bystandard procedures of organic chemistry. The preparation of suchstarting materials is described within the accompanying non-limitingExamples. Alternatively necessary starting materials are obtainable byanalogous procedures to those illustrated which are within the ordinaryskill of a chemist.

The compounds and processes of the present invention will be betterunderstood in connection with the following representative syntheticschemes that illustrate the methods by which the compounds of theinvention may be prepared, which are intended as an illustration onlyand not limiting of the scope of the invention.

Wherein n, and m each independently can be 0, 1 or 2.

EXAMPLES

The compounds and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims.

Example 1 Preparation of2-(6-bromobenzo[d][1,3]dioxol-5-ylthio)-1-(2-(neopentylamino)ethyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 1) Step 1a. 5-Bromo-6-iodobenzo[d][1,3]dioxole (Compound0102-1)

A solution of 5-bromobenzo[d][1,3]dioxole (10.0 g, 50.0 mmol), anhydrousacetonitrile (150 mL), TFA (11.4 g, 100.0 mmol) and NIS (33.7 g, 150.0mmol) was stirred at room temperature for 24 h. The solvent was removedunder reduce pressure and the crude purified by column chromatography onsilica gel (petroleum) to yield the title compound 0107-1 as a whitesolid (18.5 g, 91%): ¹H NMR (DMSO-d₆) δ 5.99 (s, 2H), 7.10 (s, 1H), 7.26(s, 1H).

Step 1b. 2-Chloro-N-(4-methoxybenzyl)-3-nitropyridin-4-amine (Compound0104)

To a solution of 2,4-dichloro-3-nitropyridine (0103) (1 g, 5.18 mmol) inDMF (8.6 mL) was added (4-methoxyphenyl)methanamine (0.71 g, 5.18 mmol)and triethylamine (0.644 mL). The solution was stirred at roomtemperature for 2 h. The mixture was evaporated to remove DMF. Theresulting mixture was purified by column chromatography on silica gel(EtOAc/petroleum at 10:1) to obtain the title compound 0104 as a yellowsolid (1.32 g, 87%): LCMS: 294 [M+1]⁺; ¹H NMR (DMSO-d₆) δ 3.72 (s, 3H),4.40 (d, 2H, J=6.3 Hz), 6.81 (d, 1H, J=5.7 Hz), 6.91 (d, 2H, J=9.0 Hz),7.25 (d, 2H, J=8.4 Hz), 7.95 (d, 1H, J=5.4 Hz), 8.02 (t, 1H, J=5.7 Hz).

Step 1c. 2-Chloro-N⁴-(4-methoxybenzyl)pyridine-3,4-diamine (Compound0105)

To a mixture of compound 0104(1.32 g, 4.49 mmol) in methanol (66 mL) andwater (6.6 mL) was added iron powder (2.51 g, 44.9 mmol) andconcentrated HCl solution (1 mL). The mixture was stirred at roomtemperature for 30 min, and then at reflux overnight. The mixture wasadjusted to pH 11 with 6N NaOH and filtered. The precipitate was washedwith methanol (10 mL). The combined filtrate and wash solution wasconcentrated and purified by column chromatography on silica gel(EtOAc/petroleum at 2:1) to obtain the title compound 0105 as a lightgreen solid (712 mg, 60%): LCMS: 264 [M+1]⁺; ¹H NMR (DMSO-d₆) δ 3.73 (s,3H), 4.31 (d, 2H, J=5.7 Hz), 4.81 (s, 2H), 6.33 (m, 2H), 6.90 (d, 2H,J=8.7 Hz), 7.26 (d, 2H, J=9.0 Hz), 7.34 (d, 1H, J=5.1 Hz).

Step 1d.4-Chloro-1-(4-methoxybenzyl)-1H-imidazo[4,5-c]pyridine-2(3H)-thione(Compound 0106)

A mixture of 0105 (2 g, 7.6 mmol), carbon disulfide (2.88 g, 37.9 mmol),potassium hydroxide (2.12 g, 37.9 mmol) in ethanol (11.5 mL) and water(1.5 mL) was heated at reflux overnight. Water (100 mL) was added afterthe mixture was allowed to cool down to room temperature. The mixturewas adjusted to pH 7 with acetic acid and then extracted with twoportions of methylene chloride. The organic layer was collected andconcentrated at reduced pressure to leave an residue which was purifiedby column chromatography on silica gel (EtOAc/petroleum at 5:1) toobtain the title compound 0106 as a white solid (2 g, 86%): LCMS: 306[M]⁺; ¹H NMR (DMSO-d₆) δ 3.68 (s, 3H), 6.41 (s, 2H), 6.86 (d, 2H, J=8.7Hz), 7.36 (m, 3H), 8.07 (d, 1H, J=5.4 Hz), 13.74 (s, 1H).

Step 1e.4-Amino-1-(4-methoxybenzyl)-1H-imidazo[4,5-c]pyridine-2(3H)-thione(Compound 0107)

A mixture of 0106 (1 g, 3.25 mmol) and sodium amide (3 g, 77 mmol) in 25mL liquid ammonia was charged in an air free sealed tube. The mixturewas then stirred at room temperature for 30 h. The mixture was cooled to−40° C. and then tube was opened. Ethanol was added carefully toterminate the reaction until no gas generated. Water (200 mL) was addedand the mixture was adjusted to pH 7 with acetic acid. The resultingsolid was filtered to obtain crude product which was purified by columnchromatography on silica gel (methylene chloride/methanol at 50:1) toobtain the title compound 0107 as a white solid (718 mg, 77%): LCMS: 287[M]⁺; ¹H NMR (DMSO-d₆) δ 3.68 (s, 3H), 5.31 (s, 2H), 6.06 (s, 2H), 6.59(d, 1H, J=6.3 Hz), 6.85 (d, 2H, J=9.0 Hz), 7.33 (d, 2H, J=8.4 Hz), 7.64(d, 1H, J=5.7 Hz), 12.53 (s, 1H).

Step 1f.2-(6-Bromobenzo[d][1,3]dioxol-5-ylthio)-1-(4-methoxybenzyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 0108-1)

A mixture of 0107 (700 mg, 2.44 mmol),5-bromo-6-iodobenzo[d][1,3]dioxole (1.20 g, 3.66 mmol), neocuproinehydrate (51 mg, 0.244 mmol), CuI (46 mg, 0.244 mmol) and NaOt-Bu (234mg, 2.44 mmol) in anhydrous DMF (31 mL) was stirred for 24 h at 110° C.(oil bath) under nitrogen atmosphere. The solvent was removed under highvacuum and the crude purified by column chromatography on silica gel(CH₂Cl₂/MeOH at 100/1) to obtain the title compound 0108-1 as a brownsolid (584 mg, 49%): LCMS: 485 [M+1]⁺; ¹H NMR (DMSO-d₆) δ 3.69 (s, 3H),5.35 (s, 2H), 6.04 (s, 2H), 6.54 (s, 1H), 6.81 (m, 4H), 7.06 (d, 2H,J=8.7 Hz), 7.29 (s, 1H).

Step 1g.2-(6-Bromobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 0109-1)

A solution of compound 0108-1 (557 mg, 1.15 mmol) and TFA (4 mL) wasstirred for 2 h at 80° C. The TFA was then evaporated and the resultingoil was adjusted to pH 7 with saturated NaHCO₃. The resultingprecipitate was collected by filtration and further purified by columnchromatography on silica gel (CH₂Cl₂/MeOH at 30/1) to give the titlecompound 0109-1 as a yellow solid (308 mg, 74%): LCMS: 365 [M+1]⁺; ¹HNMR (DMSO-d₆) δ 6.07 (s, 2H), 6.58 (s, 2H), 6.69 (d, 1H, J=6.0 Hz), 6.98(s, 1H), 7.34 (s, 1H), 7.47 (d, 1H, J=6.0 Hz).

Step 1h.2-(2-(4-Amino-2-(6-bromobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione(Compound 0111-1)

A mixture of 0109-1 (975 mg, 2.67 mmol),2-(2-bromoethyl)isoindoline-1,3-dione (1.017 g, 4.00 mmol), Cs₂CO₃(1.475 g, 4.54 mmol) in anhydrous DMF (38 mL) was stirred at 50° C. for4 h. The reaction mixture was cooled to room temperature and filtered.The filtrate was evaporated under high vacuum to give a crude product asan orange solid which was purified by column chromatography on silicagel (CH₂Cl₂/MeOH=100/1) to provide the title compound 0302-32 as a paleyellow solid.

(720 mg, 50%): LCMS: 538 [M+1]⁺.

Step 1i.1-(2-Aminoethyl)-2-(6-bromobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 0112-1)

A mixture of compound 0111-1 (720 mg, 1.337 mmol) and N₂H₄—H₂O (886 mg,14.71 mmol) in CH₂Cl₂ (27 mL) and EtOH (3 mL) was stirred at 50° C. for2 h. The solid was removed by filtration and the filtrate was washedwith brine (100 mL×2), dried over Na₂SO₄, filtered and evaporated togive the title compound 0112-1 as a pale yellow solid (495 mg, 91%):LCMS: 408 [M+1]⁺.

Step 1j.2-(6-Bromobenzo[d][1,3]dioxol-5-ylthio)-1-(2-(neopentylamino)ethyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 1)

To a solution of compound 0112-1 (150 mg, 0.613 mmol) in methanol (10mL) was added pivalaldehyde (63 mg, 0.736 mmol). After the mixture wasstirred for 30 min at room temperature, NaBH₃CN (154 mg, 2.452 mmol) wasadded slowly, and the mixture was stirred for additional 30 min. Thereaction was terminated by adding saturated NaHCO₃ (10 mL) and theresulting mixture was diluted with water (100 mL) and extracted withdichloromethane (50×2). The combined organic layer was concentrated toleave a residue which was purified by pre-HPLC to give the titlecompound 1 as a white solid (60 mg, 20%): m.p. 181-187° C. LCMS: 478[M+1]⁺; ¹H NMR (DMSO-d₆) δ 0.76 (s, 9H), 1.61 (s, 1H), 2.18 (s, 2H),2.76 (t, 2H, J=6.3 Hz), 4.24 (t, 2H, J=6.3 Hz), 6.06 (s, 2H), 6.31 (s,2H), 6.62 (s, 1H), 6.83 (d, 1H, J=5.7 Hz), 7.34 (s, 1H), 7.70 (d, 1H,J=5.7 Hz).

Example 2 Preparation of2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1-(2-(neopentylamino)ethyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 2) Step 2a. 5,6-Diiodobenzo[d][1,3]dioxole (Compound 0102-2)

A solution of 5,6-diiodobenzo[d][1,3]dioxole (1.0 g, 8.19 mmol),acetonitrile (51 mL), TFA (1.867 g) and NIS (4.05 g, 18.02 mmol) wasstirred at room temperature for 24 h. The solvent was removed under highvacuum and the crude product purified by column chromatography on silicagel (petroleum) to yield the title compound 0102-2 as a white solid(1.48 g, 48%): ¹H NMR (DMSO-d₆) δ 6.05 (s, 2H), 7.46 (s, 2H).

Step 2b.2-(6-Iodobenzo[d][1,3]dioxol-5-ylthio)-1-(4-methoxybenzyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 0108-2)

A mixture of 0107 (725 mg, 2.53 mmol), 5,6-Diiodobenzo[d][1,3]dioxole(0102-2) (1.89 g, 5.06 mmol), neocuproine hydrate (53 mg, 0.253 mmol),CuI (48 mg, 0.253 mmol) and NaOt-Bu (365 mg, 3.80 mmol) in anhydrous DMF(32 mL) was stirred for 24 h at 110° C. (oil bath) under nitrogenatmosphere. The solvent was removed under high vacuum and the crudepurified by column chromatography on silica gel (CH₂Cl₂/MeOH at 100/1)to obtain the title compound 0108-2 as a brown solid (734 mg, 55%):LCMS: 533 [M+1]⁺; ¹H NMR (DMSO-d₆) δ 3.69 (s, 3H), 5.35 (s, 2H), 6.01(s, 2H), 6.47 (s, 1H), 6.80 (d, 2H, J=9.0 Hz), 7.06 (d, 2H, J=8.7 Hz),7.41 (s, 1H).

Step 2c.2-(6-Iodobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 0109-2)

A solution of compound 0108-2 (730 mg, 1.37 mmol) in TFA (4.8 mL) wasstirred for 2 h at 80° C. The TFA was then evaporated and the resultingoil was adjusted to pH 7 with saturated NaHCO₃. The resultingprecipitate was collected by filtration and further purified by columnchromatography on silica gel (CH₂Cl₂/MeOH at 30/1) to give the titlecompound 00109-2 as a yellow solid (526 mg, 93%): LCMS: 413 [M+1]⁺; ¹HNMR (DMSO-d₆) δ 6.09 (s, 2H), 6.73 (m, 3H), 7.03 (s, 1H), 7.52 (m, 2H),12.45 (s, 1H).

Step 2d.2-(2-(4-Amino-2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione(Compound 0111-2)

A mixture of compound 0109-2 (500 mg, 1.2 mmol),2-(2-bromoethyl)isoindoline-1,3-dione (0301) (457 mg, 1.8 mmol) andCs₂CO₃ (672 mg, 2.1 mmol) in anhydrous DMF (8 mL) was stirred at 50° C.for 4 h. The reaction mixture was cooled to room temperature andfiltered. The filtrate was evaporated under high vacuum to give a crudeproduct as an orange solid which was purified by column chromatographyon silica gel (CH₂Cl₂/MeOH=100/1) to provide the title compound 0111-2as a pale yellow solid (390 mg, 56%): LC-MS: 586 [M+1]⁺. ¹H NMR (300MHz, DMSO-d₆) δ 3.92 (t, 2H, J=5.3 Hz), 4.50 (t, 2H, J=5.3 Hz), 6.00 (s,2H), 6.38 (s, 2H), 6.49 (s, 1H), 6.75 (d, 1H, J=6.0 Hz), 7.19 (s, 1H),7.64 (d, 1H, J=6.0 Hz), 7.73 (m, 4H).

Step 2e.1-(2-Aminoethyl)-2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 0112-2)

A mixture of compound 0111-2 (5 g, 8.55 mmol) and N₂H₄—H₂O (4.28 g, 85.5mmol) in CH₂Cl₂ (150 mL) and EtOH (15 mL) was stirred at 50° C. for 2 h.The solid was removed by filtration and the filtrate was washed withbrine (100 mL×2), dried over Na₂SO₄, filtered and evaporated to givecompound 32 as a white solid (3 g, 77%): m. p. 111˜121° C. LC-MS: 456[M+1]⁺. ¹H NMR (300 MHz, DMSO-d₆) δ 1.46 (s, 2H) 2.80 (t, 2H, J=6.3 Hz),4.16 (t, 2H, J=6.6 Hz), 6.05 (s, 2H), 6.29 (s, 2H), 6.69 (s, 1H), 6.84(d, 1H, J=6.0 Hz), 7.46 (s, 1H), 7.70 (d, 1H, J=5.7 Hz).

Step 2f:2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1-(2-(neopentylamino)ethyl)-1H-imidazo[4,5-c]pyridin-4-amine

The title compound 2 was prepared as a white solid (2.718 g, 26%) fromcompound 0112-2 (9.1 g, 19.9 mmol), pivalaldehyde (2.06 g, 24 mmol) andNaBH₃CN (5.027 g, 80 mmol) using a procedure similar to that describedfor compound 1 (Example 1): m.p. 203˜207° C. LCMS: 526 [M+1]⁺; ¹H NMR(DMSO-d₆) δ 0.77 (s, 9H), 1.60 (s, 1H), 2.18 (s, 2H), 2.75 (t, 2H, J=5.7Hz), 4.23 (t, 2H, J=5.4 Hz), 6.04 (s, 2H), 6.33 (s, 2H), 6.58 (s, 1H),6.83 (d, 1H, J=6.0 Hz), 7.46 (s, 1H), 7.77 (d, 1H, J=5.7 Hz).

Example 3 Preparation of2-(7-iodo-2,3-dihydrobenzo[b][1,4]dioxin-6-ylthio)-1-(2-(neopentylamino)ethyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 5) Step 3a. 6,7-Diiodo-2,3-dihydrobenzo[b][1,4]dioxine(Compound 0102-5)

To a solution of 6-iodo-2,3-dihydrobenzo[b][1,4]dioxine (2 g, 14.7 mmol)in acetonitrile (60 ml) was added NIS (9.92 g, 44.1 mmol) followed byCF₃COOH (3.35 g, 29.4 mmol). The mixture was stirred at room temperatureovernight. The reaction mixture was concentrated and purified by columnchromatography on silica gel (Petroleum ether) to provide the titlecompound 0102-5 as a white solid (0.7 g, 12%): ¹H NMR (DMSO-d₆) δ 4.21(s, 4H), 7.34 (s, 2H).

Step 3b.2-(7-Iodo-2,3-dihydrobenzo[b][1,4]dioxin-6-ylthio)-1-(4-methoxybenzyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 0108-5)

A mixture of compound 0107 (3 g, 10.5 mmol),6,7-Diiodo-2,3-dihydrobenzo[b][1,4]dioxine (0102-5) (8.1 g, 21 mmol),neocuproine hydrate (0.2 g, 1.05 mmol), CuI (0.2 g, 1.05 mmol) andNaOt-Bu (1.5 g, 15.7 mmol) in dry DMF (100 mL) was stirred at 110° C.overnight. The mixture was concentrated and purified by columnchromatography on silica gel (CH₂Cl₂/MeOH=100/1) to give the titlecompound 0108-5 as a brown solid (2.2 g, 38%): LCMS: 547[M+1]⁺; ¹H NMR(DMSO-d₆) δ 3.69 (s, 3H), 4.19 (m, 4H), 5.49 (s, 2H), 6.68 (s, 1H), 6.83(d, 2H, J=8.4 Hz), 7.11 (d, 2H, J=8.7 Hz), 7.28 (d, 1H, J=7.2 Hz), 7.35(s, 1H), 7.71 (d, 1H, J=7.2 Hz), 8.42 (s, 2H), 13.36 (s, 1H).

Step 3c.2-(7-Iodo-2,3-dihydrobenzo[b][1,4]dioxin-6-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 0109-5)

A mixture of compound 0108-5 (2.2 g, 4 mmol), trifluoroacetic acid (20mL) was stirred at reflux for 2 h. The solvent was removed and theresidue was suspended in saturated aqueous NaHCO₃ solution. Theresulting solid was collected and dried to give the title compound0206-37 as a white solid (1.5 g, 88%): LCMS: 427 [M+1]⁺; ¹H NMR(DMSO-d₆) δ 4.29 (m, 4H), 6.96 (d, 1H, J=6.9 Hz), 7.20 (s, 1H), 7.50 (s,1H), 7.63 (d, 1H, J=6.9 Hz), 8.42 (s, 2H), 13.36 (s, 1H).

Step 3d.2-(2-(4-Amino-2-(7-iodo-2,3-dihydrobenzo[b][1,4]dioxin-6-ylthio)-1H-imidazo[4,5-c]pyridin-1-yl)ethyl)isoindoline-1,3-dione(Compound 0111-5)

A mixture of compound 0109-5 (1.5 g, 3.5 mmol),2-(2-bromoethyl)isoindoline-1,3-dione (1.34 g, 5.3 mmol) and Cs₂CO₃(1.94 g, 6.0 mmol) in anhydrous DMF (50 mL) was stirred at 50° C. for 4h. The reaction mixture was cooled to room temperature and filtered. Thefiltrate was evaporated under high vacuum to give a crude product as anorange solid which was purified by column chromatography on silica gel(CH₂Cl₂/MeOH=100/1) to provide the title compound 0111-5 as a whitesolid (1.2 g, 57%): LCMS: 600 [M+1]⁺; ¹H NMR (DMSO-d₆) δ 3.92 (t, 2H,J=5.7 Hz), 4.16 (m, 4H), 4.48 (t, 2H, J=4.8 Hz), 6.38 (s, 2H), 6.41 (s,1H), 6.75 (d, 1H, J=6 Hz), 7.15 (s, 1H), 7.64 (d, 1H, J=5.7 Hz), 7.77(m, 4H).

Step 3e.1-(2-Aminoethyl)-2-(7-iodo-2,3-dihydrobenzo[b][1,4]dioxin-6-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 0112-5)

A mixture of compound 0111-5 (5 g, 8.55 mmol) and N₂H₄—H₂O (1 g, 20mmol) in CH₂Cl₂ (28 mL) and EtOH (3 mL) was stirred at 50° C. for 2 h.The solid was removed by filtration and the filtrate was washed withbrine (100 mL×2), dried over Na₂SO₄, filtered and evaporated to give thecompound 0112-5 as a yellow solid (790 mg, 84%): LC-MS: 470 [M+1]⁺. ¹HNMR (300 MHz, DMSO-d₆) δ 1.51 (m, 2H), 2.77 (t, 2H, J=6.6 Hz), 4.16 (m,6H), 6.27 (s, 2H), 6.53 (s, 1H), 6.81 (d, 1H, J=6 Hz), 7.35 (s, 1H),7.67 (d, 1H, J=5.7 Hz).

Step 3f.2-(7-Iodo-2,3-dihydrobenzo[b][1,4]dioxin-6-ylthio)-1-(2-(neopentylamino)ethyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 5)

The title compound 5 was prepared as a white solid (128 mg, 14%) fromcompound 0112-5 (790 mg, 1.7 mmol), pivalaldehyde (217 mg, 2.5 mmol) andNaBH₃CN (423 mg, 6.7 mmol) using a procedure similar to that describedfor compound 1 (Example 1): m.p. 193-200° C. LCMS: 540 [M+1]⁺; ¹H NMR(DMSO-d₆) δ 0.793 (s, 9H), 2.32 (s, 2H), 2.88 (t, 2H, J=6.3 Hz), 4.18(m, 4H), 4.32 (t, 2H, J=6.6 Hz), 6.50 (s, 1H), 6.76 (s, 2H), 6.94 (d,1H, J=6 Hz), 7.37 (s, 1H), 7.73 (d, 1H, J=6.3 Hz).

Example 4 Preparation of1-(2-(tert-butylamino)ethyl)-2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 8) Step 4a.2-(4-Amino-2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-1-yl)ethylacetate (Compound 0202-8)

A mixture of compound 0109-2 (300 mg, 0.728 mmol), 2-bromoethyl acetate(182 mg, 1.092 mmol) and Cs₂CO₃ (402 mg, 1.24 mmol) in DMF (10 mL) wasstirred at 85° C. for 2 h. DMF was evaporated under vacuum and theresidue was purified by column chromatography on silica gel (methylenechloride/methanol at 100:1) to yield the title compound 0202-8 as awhite solid (188 mg, 50.4%): LCMS: 499 [M+1]⁺; ¹H NMR (DMSO-d₆) δ 1.86(s, 3H), 4.26 (t, 2H, J=4.8 Hz), 4.45 (t, 2H, J=4.8 Hz), 6.03 (s, 2H),6.35 (s, 2H), 6.68 (s, 1H), 6.81 (d, 1H, J=6.0 Hz), 7.76 (s, 1H), 7.71(d, 1H, J=6.0 Hz).

Step 4b.2-(4-Amino-2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-1-yl)ethanol(Compound 0203-8)

A suspension of compound 0202-8 (180 mg, 0.36 mmol) in MeOH (3 mL) wastreated with K₂CO₃ (60 mg, 0.43 mmol) at 50° C. for 1 h. The mixture wasdiluted with water (15 mL) and filtered to provide the title compound0203-8 as a white solid (150 mg, 91%): LCMS: 457 [M+1]⁺; ¹H NMR(DMSO-d₆) δ 3.63 (m, 2H), 4.27 (t, 2H, J=5.4 Hz), 4.98 (t, 2H, J=5.7Hz), 6.05 (s, 2H), 6.31 (s, 2H), 6.69 (s, 1H), 6.80 (d, 1H, J=6.0 Hz),7.46 (s, 1H), 7.69 (d, 1H, J=5.7 Hz).

Step 4c.2-(4-Amino-2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-1-yl)ethylmethanesulfonate (Compound 0204-8)

Compound 0203-8 (133 mg, 0.292 mmol) was dissolved in hot anhydrousdioxane (4 mL). The solution was cooled to 40° C. and was then treatedwith NEt₃ (89 mg, 0.876 mmol) and MsCl (50 mg, 0.438 mmol) for 20 min.The mixture was concentrated and purified by column chromatography onsilica gel (CH₂Cl₂/MeOH=50/1) to provide the title compound 0204-8 as awhite solid (122 g, 78.3%): LCMS: 535 [M+1]⁺; ¹H NMR (DMSO-d₆) δ 3.07(s, 3H), 4.46 (t, 2H, J=4.5 Hz), 4.59 (t, 2H, J=5.1 Hz), 6.05 (s, 2H),6.59 (s, 2H), 6.71 (s, 1H), 6.90 (d, 1H, J=6.0 Hz), 7.48 (s, 1H), 7.73(d, 1H, J=6.6 Hz).

Step 4d.1-(2-(tert-butylamino)ethyl)-2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 8)

A solution of compound 0204-8 (250 mg, 0.47 mmol) in tert-butylamine (30mL) was stirred at 60° C. for 24 h in a pressure vessel. The solvent wasremoved and the crude was purified by column chromatography on silicagel (CH₂Cl₂/MeOH=50/1) and followed by pre-HPLC to provide the titlecompound 8 as a white solid (34 mg, 15%): m.p. 194-197° C. LCMS: 512[M+1]⁺; ¹H NMR (DMSO-d₆) δ 0.88 (s, 9H), 1.60 (s, 1H), 2.70 (t, J=6.0Hz, 2H), 4.20 (t, J=6.0 Hz, 2H), 6.05 (s, 2H), 6.35 (s, 2H), 6.70 (s,1H), 6.83 (d, J=6.0 Hz, 1H), 7.48 (s, 1H), 7.72 (d, J=6.0 Hz, 1H).

Example 5 Preparation of2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1-(2-(isopropylamino)ethyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 9)

The title compound 9 was prepared as a white solid (40 mg, 17%) fromcompound 0204-8 (252 mg, 0.47 mmol) in isopropylamine (30 mL) using aprocedure similar to that described for compound 8 (Example 4): m.p.170-172° C. LCMS: 498 [M+1]⁺; ¹H NMR (DMSO-d₆) δ 0.86 (d, J=6.3 Hz, 6H),1.68 (s, 1H), 2.62 (m, 1H), 2.74 (t, J=6.6 Hz, 2H), 4.21 (t, J=6.6 Hz,2H), 6.04 (s, 2H), 6.36 (s, 2H), 6.67 (s, 1H), 6.82 (d, J=6.0 Hz, 1H),7.47 (s, 1H), 7.71 (d, J=6.0 Hz, 1H).

Example 6 Preparation of2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1-(3-(neopentylamino)propyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 11) Step 6a.2-(3-(4-Amino-2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-1-yl)propyl)isoindoline-1,3-dione(Compound 0111-11)

The title compound 0111-11 was prepared as a pale yellow solid (410 mg,57%) from compound2-(6-Iodobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 0109-2) (500 mg, 1.2 mmol),2-(3-bromopropyl)isoindoline-1,3-dione (610 mg, 2.4 mmol) and Cs₂CO₃(652 mg, 2.0 mmol) in anhydrous DMF (8.5 mL) using a procedure similarto that described for compound 0111-2 (Example 2): LC-MS: 599.7 [M+1]⁺;¹H NMR (DMSO-d₆): δ 1.93 (m, 2H), 3.61 (t, J=6.6 Hz, 2H), 4.21 (t, J=8.1Hz, 2H), 6.04 (s, 2H), 6.40 (s, 2H), 6.50 (s, 1H), 6.87 (d, J=6.0 Hz,1H), 7.21 (s, 1H), 7.70 (d, J=6.0 Hz, 1H), 7.85 (s, 4H).

Step 6b.1-(3-Aminopropyl)-2-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 0112-11)

The title compound 0112-11 was prepared as a pale yellow solid (200 mg,74%) from compound 0111-11 (350 mg, 0.58 mmol) and N₂H₄—H₂O (580 mg,11.6 mmol) in CH₂Cl₂ (7.0 mL) and EtOH (0.6 mL) using a proceduresimilar to that described for compound 0112-2 (Example 2): LC-MS: 469.7[M+1]⁺.

Step 6c.2-(6-Iodobenzo[d][1,3]dioxol-5-ylthio)-1-(3-(neopentylamino)propyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 11)

The title compound 11 was prepared as a white solid (110 mg, 37%) fromcompound 0112-11 (257 mg, 0.55 mmol) and pivalaldehyde (60 mg, 0.70mmol). using a procedure similar to that described for compound 1(Example 1): m.p. 170-174° C. LCMS: 540 [M+1]⁺; ¹H NMR (DMSO-d₆): δ 0.84(s, 9H), 1.76 (m, 2H), 2.14 (s, 2H), 2.43 (t, J=6.9 Hz, 2H), 4.23 (t,J=7.2 Hz, 2H), 6.04 (s, 2H), 6.36 (s, 2H), 6.63 (s, 1H), 6.80 (d, J=6.0Hz, 1H), 7.47 (s, 1H), 7.71 (d, J=6.0 Hz, 1H).

Example 7 Preparation of2-(6-(dimethylamino)benzo[d][1,3]dioxol-5-ylthio)-1-(2-(neopentylamino)ethyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 15) Step 7a. 6-Iodo-N,N-dimethylbenzo[d][1,3]dioxol-5-amine(Compound 0102-15)

To a solution of 3,4-(Methylenedioxy)aniline (8 g, 58.3 mmol) in AcOH(120 ml) was added Ac2O (48 mL). The mixture was stirred for overnight.After reaction, the mixture was poured into saturated NaHCO₃ solution,and then filtered. The filtrate was extracted with ethyl acetate to giveN-(benzo[d][1,3]dioxol-5-yl)acetamide (10 g, 95%). LCMS: 180[M+1]; ¹HNMR (DMSO-d⁶) δ 2.0 (s, 3H), 5.96 (s, 2H), 6.82 (d, 1H, J=8.1 Hz), 6.91(d, 1H, J=2.1 Hz), 7.30 (d, 1H, J=1.8 Hz), 9.84 (s, 1H).

A 1.0 M solution of iodine monochloride in methylene chloride (72.6 mL)was added dropwise to a solution ofN-(benzo[d][1,3]dioxol-5-yl)acetamide (10 g, 55.8 mmol) in methylenechloride (66 mL) and acetic acid (11 mL). The mixture was stirred undernitrogen overnight and then washed with saturated sodium thiosulfate(2×150 mL) and brine (150 mL). The methylene chloride solution was dried(MgSO₄) and evaporated, and the residue was purified by columnchromatography on silica gel (CH₂Cl₂/petroleum at 20/1) to obtainN-(6-iodobenzo[d][1,3]dioxol-5-yl)acetamide (3.7 g, 22%) as a whitesolid. LCMS: 306 [M+1]⁺; ¹H NMR (DMSO-d⁶) δ 2.00 (s, 3H), 6.06 (s, 2H),6.95 (s, 1H), 7.37 (s, 1H), 9.34 (s, 1H).

A solution of N-(6-iodobenzo[d][1,3]dioxol-5-yl)acetamide (200 mg, 0.656mmol) and NaOH (1.31 g, 32.8 mmol) in ethanol (26 mL) and water (6 mL)was heated to reflux with stirring for 4 h. The mixture was cooled andthe solvent was removed under vacuum. The residue was partitionedbetween methylene chloride (100 mL) and water (100 mL). The organiclayer was washed with water (2×100 mL), dried (MgSO₄) and evaporatedunder vacuum to give 6-iodobenzo[d][1,3]dioxol-5-amine (170 mg, 98%) asorange solid. LCMS: 264 [M+1]⁺; ¹H NMR (DMSO-d⁶) δ 4.88 (s, 2H), 5.87(s, 2H), 6.47 (s, 1H), 7.07 (s, 1H).

To a solution of 6-iodobenzo[d][1,3]dioxol-5-amine (1 g, 3.8 mmol) andparaformaldehyde (1.14 g, 38 mmol) in methanol (10 mL) was added NaBH₃CN(2.39 g, 38 mmol) slowly with stirring. The mixture was heated to 50° C.for 4 h. Water (100 mL) was added and extracted with methylene chloride(100 mL). The organic layer was washed with brine (100 mL), dried(MgSO₄) and evaporated under vacuum to give crude title compound 0102-15(1.16 g) as a brown oil which was used directly to the next step withoutfurther purification. LCMS: 292 [M+1]⁺; ¹H NMR (DMSO-d⁶) δ 2.56 (s, 6H),6.02 (s, 2H), 6.96 (s, 1H), 7.32 (s, 1H).

Step 7b. tert-Butyl 2-(2-chloro-3-nitropyridin-4-ylamino)ethylcarbamate(Compound 0301-15)

A mixture of 2,4-dichloro-3-nitropyridine (0103) (55 g, 0.285 mol),tert-butyl N-(2-aminoethyl)carbamate (59.3 g, 0.37 mol) and Et₃N (43.2g, 0.427 mol) in DMF (450 mL) was heated to 65° C. and stirred for 2.5h. The DMF was removed under reduced pressure and the residue was pouredinto brine, extracted with EtOAc, dried and concentrated. The residuewas then recrystallized with EtOH-water to provide the title compound0301-15 (65 g, 72%) as a yellow solid: LCMS: 317 [M+1]⁺; ¹H NMR(DMSO-d⁶) δ 1.36 (s, 9H), 3.10 (q, 2H, J₁=8.0 Hz, J₂=16 Hz), 3.30 (q,2H, J, =8.0 Hz, J₂=16 Hz), 6.98 (d, 2H, J=8 Hz), 7.38 (t, 1H, J=7.2 Hz),8.04 (d, 1H, J=8.0 Hz).

Step 7c. tert-Butyl 2-(3-amino-2-chloropyridin-4-ylamino)ethylcarbamate(Compound 0302-15)

A mixture of compound 0301-15 (70 g, 0.221 mol), iron dust (62 g, 1.105mol) and FeSO₄7H₂O (18.5 g, 66 mmol) in saturated NH₄Cl aqueous solution(750 mL) and MeOH (1400 mL) was heated to 80° C. for 3 h. The reactionwas then filtered and washed with MeOH. The filtrate was concentratedand the residue was dissolved in dichloromethane. The dichloromethanesolution was washed with water and concentrated to give the titlecompound 0302-15 (55 g, 87%) as a red solid. LCMS: 287 [M+1]⁺; ¹H NMR(DMSO-d⁶) δ 1.37 (s, 9H), 3.13 (m, 4H), 4.69 (s, 2H), 5.76 (d, 1H, J=5.2Hz), 6.45 (d, 1H, J=5.6 Hz), 6.92 (d, 1H, J=5.2 Hz), 7.41 (d, 1H, J=5.2Hz).

Step 7d. tert-Butyl2-(4-chloro-2-thioxo-2,3-dihydroimidazo[4,5-c]pyridin-1-yl)ethylcarbamate(Compound 0303-15)

A mixture of compound 0302-15 (55 g, 0.192 mol), KOH (54 g, 0.959 mol),CS₂ (73 g, 0.959 mol) in EtOH (500 mL) and H₂O (50 mL) was stirred for12 h at 85° C. Then the mixture was cooled to room temperature anddiluted with water. The mixture was adjusted to pH 7 with AcOH, filteredto give the title compound 0303-15 (54.5 g, 87%) as a yellow solid:LCMS: 329 [M+1]⁺; ¹H NMR (DMSO-d⁶) δ 1.20 (s, 9H), 3.33 (s, 2H), 4.24(t, 2H, J=4.8 Hz), 6.89 (t, 1H, J=5.2 Hz), 7.33 (d, 1H, J=5.2 Hz), 8.14(d, 1H, J=5.2 Hz), 13.59 (s, 1H).

Step 7e.1-(2-Aminoethyl)-4-chloro-1H-imidazo[4,5-c]pyridine-2(3H)-thione salt(Compound 0304-15)

A mixture of compound 0303-15 (63.8 g, 0.194 mol) and TFA (150 mL, 1.94mol) in dichloromethane (750 mL) was stirred for 2 h at 25° C. Thesolvent was removed and dried to give the title compound 0304-15 (163 g)as a yellow solid which was used directly in next step without furtherpurification: LCMS: 229 [M+1]⁺; ¹H NMR (DMSO-d⁶) δ 3.27 (q, 2H, J=5.2Hz, J₂=11.2 Hz), 4.47 (t, 2H, J=6.0 Hz), 7.55 (d, 1H, J=5.2 Hz), 7.92(s, 2H), 8.20 (d, 1H, J=5.2 Hz), 12.22 (s, 2H), 13.78 (s, 1H).

Step 7f.4-Chloro-1-(2-(neopentylamino)ethyl)-1H-imidazo[4,5-c]pyridine-2(3H)-thione(Compound 0305-15)

A suspension of compound 0304-15 (163 g, 0.194 mol) in MeOH (1300 mL)was adjusted to pH 8 with NEt₃ (˜100 mL) at ice bath. Then pivalaldehyde(33.4 g, 0.388 mol) was added to the mixture and the mixture was stirredfor 30 min at room temperature. NaBH₃CN (48.76 g, 0.776 mol) was addedto the mixture and the mixture was stirred at room temperatureovernight. The resulting solid was filtered to give the title compound0305-15 (38.6 g, total yield of two steps: 67%) as a yellow solid: LCMS:299 [M+1]⁺; ¹H NMR (DMSO-d⁶) δ 0.79 (s, H), 2.36 (s, 2H) 2.95 (t, 2H,J=6.0 Hz), 4.32 (t, 2H, J=6.0 Hz), 7.49 (d, 1H, J=5.6 Hz), 8.07 (d, 1H,J=5.6 Hz).

Step 7g.4-Amino-1-(2-(neopentylamino)ethyl)-1H-imidazo[4,5-c]pyridine-2(3H)-thione(Compound 0306-15)

A mixture of compound 0305-15 (11.4 g, 38.2 mmol) and sodium amide (30g, 769 mmol) in 400 mL liquid ammonia was stirred at 25° C. for 24 h ina autoclave. Ammonia was volatilized before opening the autoclave. Waterwas added carefully until all solids were dissolved. This solution wasadjust pH 7 with acetic acid and filtered to obtain the title compound0306-15 (9 g, 84%) as a gray solid: LCMS: 280 [M+1]⁺; ¹H NMR (DMSO-d⁶)(0.792 (s, 9H), 2.27 (s, 2H), 2.84 (m, 2H), 4.19 (m, 2H), 6.06 (s, 2H),6.77 (m, 1H), 7.71 (m, 1H).

Step 7h.2-(6-(Dimethylamino)benzo[d][1,3]dioxol-5-ylthio)-1-(2-(neopentylamino)ethyl)-1H-imidazo[4,5-c]pyridin-4-amine(Compound 15)

A mixture of 0306-15 (100 mg, 0.36 mmol), 0102-15 (200 mg, 0.68 mmol),neocuproine hydrate (14 mg, 0.068 mmol), CuI (12 mg, 0.068 mmol) andNaOt-Bu (66 mg, 0.068 mmol) in anhydrous DMF (6 mL) was stirred for 12 hat 110° C. (oil bath) under nitrogen atmosphere. The solvent was pouredinto water, the mixture was then extracted with ethyl acetate. Solventswere removed and the crude was purified by prep-TLC (CH₂Cl₂/MeOH at20/1) to obtain the title compound 15: LCMS: 443 [M+1]⁺; ¹H NMR(DMSO-d⁶) δ 0.748 (s, 9H), 2.16 (s, 2H), 2.63 (s, 6H), 2.74 (t, 2H,J=6.0 Hz), 4.21 (t, 2H, J=6.0 Hz) 5.94 (s, 2H), 6.16 (s, 1H), 6.38 (s,2H), 6.84 (d, 1H, J=6.0 Hz), 7.01 (s, 1H), 7.70 (d, 1H, J=5.6 Hz).

Biological Assays:

As stated hereinbefore the derivatives defined in the present inventionpossess anti-proliferation activity. These properties may be assessed,for example, using one or more of the procedures set out below:

(a) An In Vitro Assay which Determines the Ability of a Test Compound toInhibit Hsp90 Chaperone Activity.

The Hsp90 chaperone assay was performed to measure the ability of HSP90protein to refold the heat-denatured luciferase protein. HSP90 was firstincubated with different concentrations of test compounds indenaturation buffer (25 mM Tris, pH7.5, 8 mM MgSO₄, 0.01% bovine gammaglobulin and 10% glycerol) at room temperature for 30 min. Luciferaseprotein was added to denaturation mix and incubated at 50° C. for 8 min.The final concentration of HSP90 and luciferase in denaturation mixturewere 0.375 μM and 0.125 μM respectively. A 5 μl sample of the denaturedmix was diluted into 25 μl of renaturation buffer (25 mM Tris, pH7.5, 8mM MgSO4, 0.01% bovine gamma globulin and 10% glycerol, 0.5 mM ATP, 2 mMDTT, 5 mM KCl, 0.3 μM HSP70 and 0.15 μM HSP40). The renaturationreaction was incubated at room temperature for 150 min, followed bydilution of 10 μl of the renatured sample into 90 μl of luciferinreagent (Luclite, PerkinElmer Life Science). The mixture was incubatedat dark for 5 min before reading the luminescence signal on a TopCountplate reader (PerkinElmer Life Science).

(b) HSP90 Competition Binding (Fluorescence Polarization) Assay.

A fluorescein isothiocyanate (FITC) labeled GM was purchase fromInvivoGen (ant-fgl-1). The interaction between HSP90 and labeled GMforms the basis for the fluorescence polarization assay. A free andfast-tumbling FITC labeled GM emits random light with respect to theplane of polarization plane of excited light, resulting in a lowerpolarization degree (mP) value. When GM is bound to HSP90, the complextumble slower and the emitted light is polarized, resulting in a highermP value. This competition binding assay was performed in 96-well plateand with each assay contained 10 and 50 nM of labeled GM and purifiedHSP90 protein (Assay Design, SPP-776F) respectively. The assay buffercontained 20 mM HEPES (pH 7.3), 50 mM KCl, 1 mM DTT, 50 mM MgCl₂, 20 mMNa₂MoO₄, 0.01% NP40 with 0.1 mg/ml bovine gamma-globulin. Compounds arediluted in DMSO and added to the final assay before labeled GM withconcentration range from 20 uM to 2 nM. mP value was determined byBioTek Synergy II with background subtraction after 24 hours ofincubation at 4° C.

The following TABLE B lists compounds representative of the inventionand their activity in HSP90 assays. In these assays, the followinggrading was used: I≧10 μM, 10 μM>II>1 μM, 1 μM>III>0.1 μM, and IV≦0.1 μMfor IC₅₀.

TABLE B HSP90 Chaperone HSP90 Binding Compound No. (IC₅₀) (IC₅₀) 1 III 2III IV 3 III 5 IV 8 III 9 III 10 III 11 III 15 IVPK in Mouse Blood and Tissues after Oral and Intravenous Administration:

Female athymic nude (CD-1 nu/nu) were obtained from Charles RiverLaboratories (Wilmington, Mass.). They were housed in ventilatedmicro-isolator cages in Curis animal facility conditioned at temperatureof 23±1° C., humidity of 50-70%, and a 12-hour light/12-hour dark cycle.The animals were fed with irradiated laboratory rodent diet ad libitumand provided with sterilized water. HCT116 human colorectal cancer cellswere cultured in RPMI-1640 medium plus 5% FBS (fetal bovine serum) in 5%CO₂ incubator. When the cells reached about 70-90% confluent, they wereharvested by treatment with trypsin-EDTA (0.25% Trypsin, 1 mM EDTA). Thecell pellet was diluted in HBSS (Hank's balanced salt solution) forimplantation. For subcutaneous (s.c) tumor implantation, 5 million ofcells were injected into the right hind flank region of each mouse. Whentumors reached around 200 mm³, mice were dosed orally with a solutioncontaining compounds that need to be evaluated. The concentration foreach compound in the solution was kept below 5 mg/ml. The maximum numberof compound in one cassette was 6. 30% Captisol (Cydex) was used for allformulation. At various time points (3 mice per time point) followingcompound administration, mice were euthanized with CO₂, blood andtissues were collected. Blood was collected into tubes containing sodiumheparin. The plasma was separated via centrifugation and stored at −40°C. before analysis. Tissues including tumors were homogenized in 0.8 mlwater. An internal standard was added into the tissue homogenates. Thehomogenates were extracted with 1 ml ethyl acetate for three times.After evaporation, the residual was reconstituted in 0.1 ml acetonitrilefor LC/MS/MS assay (Agilent HPLC 1100 Series). FIGS. 1-3 shows thetissue concentration and pharmacokinetic profiles from cassette dosestudies of compounds 2 and 15 as determined through additional studies.

TABLE C HSP90 Compounds in Mouse Brain (ng/g) after i.v. delivery (5mg/g Cassette Study) Time (hr) 21 2 20 15 0.08333 4076.8 4405.4s 588.43236.8 0.5 1913.2 2510.2 327.5 1449.8 2 968.4 1323.0 252.4 773.3 6 245.9392.0 67.6 177 24 39.7 47.9 6.7 27.0 48 6.0 0.1 2.3 4.8

TABLE D HSP90 Compounds in Mouse Brain (ng/g) after oral delivery (10mg/g Cassette Study) Time (hr) 21 2 20 15 0.5 108.9 148.0 22.2 61.2 2409.0 792.7 74.9 255.6 6 373.6 833.9 83.9 217.9 24 10.8 8.2 4.0 5.9 481.4 1.1 0.8 0.7

TABLE E AUC values of HSP90 compounds in the brain Compound No. 21 2 2015 AUC i.v. (hr*ng/g) 8956.3 12281.1 2041.8 6761.3 AUC p.o. (hr*ng/g)5587.4 11686.8 1243.7 3293.9 F (%) 31.2% 47.6% 30.5% 24.4%

PK profile of compound-15 was measured after oral administration of 30mg/kg dose in a single dose study. A T_(1/2) of 3.30 hours, a T_(max) of2.00 hours, a C_(max) of 1378 ng/mL, an AUC₀₋₂₄ of 10414 hr*ng/mL, andan AUC_(inf) of 10480 hr*ng/mL were measured. FIGS. 4-7 show theconcentrations and pharmacokinetic profiles of compound 15 in plasma,tumor, brain and lung tissues from separate studies.

A similar procedure as above was performed in nude mice with U87MGXenografts. The study showed good to excellent results for the crossingof all compounds tested with superior results observed for Compound 15(Table F). A similar procedure as above with U87MG Xenografts were usedto study the pharmacodynamic effects of 40 mg/Kg, 80 mg/Kg and 160 mg/Kgoral dosage (every other day) of Compound-15. (FIGS. 21A and B). FIG.21B shows results analyzed by Western blot in U87MG s.c. tumors treatedwith Compound-15 for 3 weeks compared with vehicle control (n=4). Aftera dosing period of 21 days (40, 80 or 160 mg/kg every other day), micewere sacrificed and 4 tumors from each group were collected for Westernblot analysis using antibodies as indicated. Results showeddose-dependent inhibition of multiple HSP90 client proteins, correlatingwith efficacy results. (FIG. 21B)

TABLE F HSP90 Compounds in Mouse Brain (ng/g) after oral (30 mg/Kg)delivery Time (hr) 22 15 20 23 0 0 0 0 0 0.5 2.5 66.4 29.3 5.4 1 2.2124.1 56.6 13.6 3 3.7 257.1 109.3 21.5 6 0.9 77.8 37.9 9.1 24 1.3 3.21.8 3.0 AUC (hr*ng/g) 36.7 1692.2 782.7 231.8

Blood Brain Barrier Penetration and c log P Values:

The octanol/water partition coefficient (c Log P) values for severalcompounds of the invention were calculated using ACD/ChemSketch programavailable from Advanced Chemistry Development Inc. (Table G). Thecalculated c Log P values showed a correlation to effective crossing ofthe blood brain barrier. Based on the results, compounds of formula Iwith a c log P value of about 3.70 or more are considered effective incrossing the blood brain barrier. Preferred compounds have a c Log Pvalue of at least 4.00 and most preferred compounds have a c Log P valueof at least 4.20.

TABLE G cLogP values Compound No. cLogP 2 5.59 15 4.32 20 3.46 21 4.8522 3.24 23 2.98

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

Activity of Compound-15 Against Erlotinib-Resistant NSCLC

H1993 and H1975 NSCLC cells were incubated with 1 mol/L of Compund-15for 7 hours and cultured in compound-free medium for an additional 0, 17or 24 hours and analyzed by Western blot. (Results are shown in FIGS.15A and B). Compound-15 treatment reduced the levels ofphosphorylated-MET as well as total MET, the amplification of which isresponsible for erlotinib resistance in NSCLC. In addition, Compound-15treatment suppressed downstream PI3K/AKT and RAF/ERK signaling as shownby reduced p-AKT/AKT and p-ERK/ERK levels (FIGS. 15A and B).

HSP90/Compound-15 Binding Assay:

The ability of compound-15 to interact with HSP90 derived fromerlotinib-resistant NSCLC cells, fluorescence polarization assay wasconducted with geldanamycin competition using cell extracts preparedfrom cultured H1975 and H1993 NSCLC cell lines, which become resistantto erlotinib due to EGFRT790M mutation and c-MET amplification,respectively. Compound 15 strongly bind to cancer derived HSP90 complexwith an IC₅₀ of 61.2 nmol/L in H1975 and 74.2 nmol/L in H1993,respectively (FIG. 16).

Inhibition of HSP90 Client Proteins by Compound-15:

A pharmacodynamic study in H1975 subcutaneous tumors showed potentinhibition of multiple HSP90 client proteins and induction of apoptosisfollowing a single dose of Compound-15 at 160 mg/kg (FIG. 17). Mostimportantly, the compound induced degradation of mutant EGFR, the geneconferring oncogenicity and erlotinib resistance in the H1975 cell line.The degradation of EGFR was accompanied by inhibition of its downstreamsignaling molecules of the cell proliferation (RAF, p-ERK) and survival(p-AKT) pathways, with concurrent induction of HSP70, a marker of HSP90inhibition. Furthermore, Compound-15 robustly induced apoptosis at boththe 6- and 24-hour time points as measured by PARP and caspase-3cleavages.

Efficacy Study in the H1975 Subcutaneous Tumor Model:

Cells (3.5×106) were implanted subcutaneously into nude mice. Treatmentwith vehicle control or Compound-15 at 80, 120, or 160 mg/kg (orally,once every 2 days) started when tumors reached an average volume of 118mm³. Dose-dependent inhibition of tumor growth was observed (FIG. 18A),without significant loss of body weight (FIG. 18B).

Efficacy Study in the H1975 Orthotopic Lung Tumor (NSCLC) Model:

H1975 cells (2×106) were implanted orthotopically to the left lung ofnude mice. Starting four days after tumor implantation, mice weretreated with Compound-15 (120 mg/kg, orally, once every two days),erlotinib (50 mg/kg, orally, once a day), or vehicle control.Compound-15 treatment for 5 wk significantly prolonged animal survival(P=0.001), whereas erlotinib displayed no therapeutic benefit (P>0.05)(FIG. 19A).

Dose-dependent efficacy study of Compound-15 in H1975 orthotopic lungtumor model (n=11): Mice were treated with Compound-15 (20, 40, 80, and120 mg/kg) starting 4 day after tumor implantation. Compound-15dose-dependently extended animal survival following a 5-wk dosingregimen (FIG. 19B).

Pharmacodynamic Study in H1975 Orthotopic Lung Tumors:

Animals bearing H1975 orthotopic lung tumors were treated withCompound-15 at 160 mg/kg once. Tumors were collected at various timepoints (n=2-3) and subjected to Western blot analysis. Sustainedinduction of HSP70 and apoptosis and inhibition of oncoproteinphosphorylated-AKT were observed (FIG. 19C).

Efficacy Study in H1975 Intracranial Metastasis Model (n=10):

H1975 cells (5×105) were implanted intracranially into nude mice.Starting 5 days after tumor implantation, mice were treated withCompound-15 (120 mg/kg, orally, once every two days), lapatinib (75mg/kg, orally, twice a day), or vehicle control. Compound-15 treatmentfor 4 wk significantly prolonged animal survival (P=0.001) in contrastto the lack of lapatinib efficacy (P>0.05) (FIG. 19D).

Efficacy Study in Rat tMCAO Model:

Male Wistar rats were subjected to 90 min tMCAO followed byadministration of single dose of Compound-2 at 4 hrs post-tMCAO onset byIV. Rats were euthanized 48 hrs post-tMCAO and size of infarct wasanalyzed by TTC staining (FIGS. 11 and 12).

Induction of HSP70 Up-Regulation in Primary Hippocampal Neuron Cultures:

Hippocampal neurons were cultured from E17 rats. Cultures at DIV 5 weretreated with Compound-21 for 24 hrs. Compound-21 induced HSP70 levelincrease (FIG. 13).

Effect of Compound-21 on PHF-tau Level in primary hippocampal neuroncultures: High endogenous PHF-tau level is due to the nature ofembryonic culture Compound-21 and Aβ oligomer were co-administrated tothe culture at DIV 5. Aβ oligomer treatment induces slight increase ofPHF-tau level. Compound-21 significantly decreases the PHF-tau level(FIG. 14).

Pharmacodynamic Study in K-Ras-Mutated A549 (Human Lung AdenocarcinomaEpithelial Cell Line) Subcutaneous Tumors:

Compound-15 was dosed at 160 mg/kg, and tumors were collected at the 6-and 24-h time points (n=4) and subjected to Western blot analysis usingantibodies as indicated. Potent inhibition of HER2 as well as keycomponents of the PI3K/AKT and RAF/MEK/ERK signaling pathways, alongwith concurrent induction of HSP70 at both the 6- and 24-h time points,was observed (FIG. 20A).

Efficacy Study was Conducted in A549 Subcutaneous Tumor Model (n=7).

Cells (5×106) were implanted subcutaneously into nude mice. Treatmentwith vehicle or Compound-15 (160 mg/kg, orally, once every two days)started when tumors reached an average volume of 84 mm³. Tumor stasiswas observed in the treatment group (T/C 9.5%, P<0.001). Tumor volumeswere expressed as mean±SE. (FIG. 20B) Efficacy study was conducted inA549 orthotopic lung tumor model (n=10). A549 cells (1×106) wereimplanted orthotopically to the left lung of nude mice. Beginning 4 dafter tumor implantation, mice were treated with Compound-15 (120 mg/kg,orally, once every two days), erlotinib (50 mg/kg, orally, once a day),or vehicle control for 4 wk. Compound-15 treatment significantlyprolonged animal survival (P=0.001), whereas erlotinib exhibited noantitumor effect (P>0.05) (FIG. 20C). Efficacy study of Compound-15 wasconducted in combination with paclitaxel in H1975 subcutaneous tumormodel (n=9). H1975 cells (4×106) were implanted subcutaneously into nudemice. When tumors reached an average volume of 150 mm³, animals weretreated with either Compound-15 (160 mg/kg, orally, once every twodays), paclitaxel (12.5 mg/kg, i.p., twice weekly), or a combination ofthe two agents. An enhanced antitumor effect was observed in thecombination group (P<0.05) (FIG. 20 D).

Efficacy Study of Compound-15 in MV4-11 (Human Lymphoblast Cell Line)s.c. Tumors:

After a single oral dosing of Compound-15 at 160 mg/kg, tumors werecollected at 6 and 24 h (n=3), homogenized, and analyzed by Western blot(FIG. 22A). In an efficacy study using MV4-11 s.c. tumor model (n=8),MV4-11 AML tumor cells (20×10⁶) were implanted s.c. into sever combinedimmunodeficient mice. Treatment with Compound-15 (160 mg/kg, orally,q2d) started when tumors reached an average volume of 146 mm³.Compound-15 treatment for three weeks, q2d (every other day), showedcomplete tumor regression (FIG. 22B). Efficacy study was conducted inlarger mice with larger sized tumors, with pretreatment volume of 380and 835 mm³, FIGS. 22C and 22D respectively.

PD Study after Single Oral Dosing of Compound-15 in H1975 (NSCLC) s.c.Tumors:

After a single oral dosing of CUDC-305 at 80 mg/kg, tumors werecollected at 8 h (n=3), homogenized, and analyzed by Western blot usingantibodies as indicated. EGFR and multiple signaling molecules wereinhibited (FIG. 23A).

Efficacy Study in the H1975 s.c. Tumor Model (n=9):

H1975 NSCLC cells (5×10⁶) were implanted s.c. into nude mice. Treatmentwith Compound-15 (160 mg/kg, orally, q2d) started when tumors reached anaverage volume of 160 mm³. Dosing of Compound-15 for 3 wk significantlyinhibited tumor growth compared with vehicle control (T/C, 15.4%;P<0.001) (FIG. 23B).

Efficacy Study in the MDA-MB-468 Orthotopic Tumor Model (n=8):

MDA-MB-468 breast cancer cells (20×10⁶) were implanted orthotopicallyinto nude mice. Treatment started when tumor size reached an averagevolume of 113 mm³. Compound-15 was dosed at 120 mg/kg orally q2d;paclitaxel was dosed at 12.5 mg/kg i.p. twice weekly. Compound-15delivered as a single agent induced tumor regression by 3.4% (P<0.001).An enhanced antitumor effect was observed when Compound-15 was combinedwith paclitaxel (tumor regression of 36.6%, P<0.001) (FIG. 23C).

Efficacy Study in the Colo205 s.c. Tumor Model (n=10):

Colo205 colorectal cancer cells (5×10⁶) were implanted s.c. into nudemice. Treatment with Compound-15 started when tumors reached an averagevolume of 120 mm³. Compound-15 was dosed at 120 mg/kg orally q2d.Camptothecin-11 was dosed at 15 mg/kg i.p. twice weekly. Compound-15delivered as single agent significantly inhibited tumor growth (T/C,14.2%; P<0.001). However, an enhanced antitumor activity was observed inthe group treated with Compound-15 and Camptothecin-11 combination(P<0.05) (FIG. 23D). Brain penetration activity of Compounds 2 and 21was compared to reference compounds VER-052296, Cmp42, SNX-2112 that areknown for HSP90 inhibitory activity (FIGS. 9 and 10).

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of treating lung cancer comprising theoral administration of a compound of formula I:

or its pharmaceutically acceptable salts thereof, wherein; n is 1 or 2;R₁ and R₂ are independently H, C₁-C₈ alkyl, or C₁-C₈ substituted alkyl;R₃ and R₄ are independently H, C₁-C₈ alkyl, or C₁-C₈ substituted alkyl;R₅ is halogen, —SR₆ or —NR₆R₇ wherein R₆ and R₇ are independently H,C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₂-C₈ alkenyl, C₂-C₈ substitutedalkenyl, C₂-C₈ alkynyl, C₂-C₈ substituted alkynyl or C₃-C₈ cycloalkyl;and, Y is C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₂-C₈ alkenyl, C₂-C₈substituted alkenyl, C₂-C₈ alkynyl, C₂-C₈ substituted alkynyl, C₃-C₈cycloalkyl or C₃-C₈ substituted cycloalkyl.
 2. The method of claim 1,wherein the lung cancer is small cell lung cancer.
 3. The method ofclaim 1, wherein the lung cancer is non-small cell lung cancer.
 4. Themethod of claim 1, wherein said non-small cell lung cancer is selectedfrom adenocarcinoma, squamous cell lung carcinoma and large cell lungcarcinoma.
 5. A method according to claim 1, wherein said compound offormula I has a c Log P value of more than 3.70.
 6. A method accordingto claim 5, wherein said c Log P value is more than about 4.00.
 7. Amethod according to claim 6, wherein said c Log P value is more thanabout 4.20.
 8. A method of treating lung cancer in a patient that failsto respond to treatment by an inhibitor of epidermal growth factor(EGFR) selected from gefitinib, erlotinib, vandetanib, AEE-788, PKI-166,PTK787/ZK222584, lapatinib, cetuximab, nimotuzumab, matuzumab,panitumumab, trastuzumab and pertuzumab by administration of a compoundof formula I.
 9. The method of claim 8, wherein said patient harbors amutation to the EGFR gene.
 10. The method of claim 8, wherein saidpatient has acquired resistance to EGFR inhibitors.
 11. The method claim10, wherein said acquired resistance results from a T790M mutation ofthe EGFR gene.
 12. The method of claim 10, wherein said acquiredresistance results from D761Y mutation of the EGFR gene.
 13. The methodof claim 10, wherein said acquired resistance results from L858Rmutation of the EGFR gene.
 14. The method of claim 8, wherein saidpatient harbors a mutation to K-Ras gene.
 15. The method of claim 14,wherein said mutation renders the patient resistant to treatment byerlotinib and/or lapatinib.
 16. A method according to claim 1, whereinthe compound is selected from:

or pharmaceutically acceptable salts thereof.
 17. A method of regulatingthe level of HSP70 in the brain tissue of patient by the oraladministration of a compound of formula I to a patient in need thereof.18. The method according to claim 17, wherein said level of HSP70 isincreased.